CREB (D76D11) Rabbit mAb #4820

Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween^® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH₂O, mix.
3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH₂O.
4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH₂O, mix.
5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH₂O, mix.
6. 10X Tris Buffered Saline with Tween^® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH₂O, mix.
7. Nonfat Dry Milk: (#9999).
8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
9. Wash Buffer: (#9997) 1X TBST.
10. Bovine Serum Albumin (BSA): (#9998).
11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
12. Biotinylated Protein Ladder Detection Pack: (#7727).
13. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

1. Treat cells by adding fresh media containing regulator for desired time.
2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
6. Microcentrifuge for 5 min.

7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

   NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm²) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with 15 ml of TBST.
3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with 15 ml of TBST.
5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH₂O, mix.

2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH₂O, mix.

   NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
4. Protein A Agarose Beads: (#9863).
5. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH₂O, mix.
6. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate on ice three times for 5 sec each.
6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

1. Vortex to mix beads.
2. Add 10–30 µl of 50% Protein A agarose bead slurry to 200 µl cell lysate at 1 mg/ml.
3. Incubate with rotation at 4°C for 30–60 min.
4. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
5. Proceed to immunoprecipitation below.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.

1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
2. Add protein A agarose (10–30 µl of 50% bead slurry). Incubate with rotation for 1–3 hr at 4°C.
3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
4. Proceed to sample analysis by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.

When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.

For Analysis by Kinase Assay

1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
3. Incubate for 30 min at 30°C.
4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
5. Transfer supernatant containing phosphorylated substrate to another tube.
6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).

Immunofluorescence (Frozen)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix. Adjust pH to 8.0.
2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
3. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.

5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414

   NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

6. Prolong^® Gold AntiFade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section C).
2. For fresh, unfixed frozen tissue, please fix immediately, as follows:

   1. Cover sections with 4% formaldehyde dilute in 1X PBS.

      NOTE: Formaldehyde is toxic, use only in fume hood.

   2. Allow sections to fix for 15 minutes at room temperature.
   3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes each.
   4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in Blocking Buffer for 60 min.
2. While blocking, prepare primary antibody by diluting as indicated on protocol on product webpage in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Coverslip slides with Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold Antifade Reagent with DAPI (#8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix. Adjust pH to 8.0.
2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH₂O, mix well. While stirring, add 30 µl Triton™ X-100.
4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.

5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414
6. Prolong^® Gold AntiFade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

   NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in Blocking Buffer for 60 min.
2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Coverslip slides with Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold Antifade Reagent with DAPI (#8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Flow Cytometry, Methanol Permeabilization Protocol for Rabbit Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
2. 4% Formaldehyde, Methanol-Free (#47746)
3. 100% Methanol (#13604): Chill before use
4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
5. Recommended Anti-Rabbit secondary antibodies::
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 647 Conjugate) #4414
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

1. Pellet cells by centrifugation and remove supernatant.
2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
3. Fix for 15 min at room temperature (20-25°C).
4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
   1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
2. Permeabilize for a minimum of 10 min on ice.
3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells per assay.)
2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
4. Incubate for 1 hr at room temperature.
5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
7. Incubate for 30 min at room temperature. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

CUT&Tag Protocol

I. Activation of Concanavalin A Beads

Before Starting:

! All buffer volumes should be scaled proportionally to the number of CUT&Tag reactions being performed.

• Place Concanavalin A Bead Activation Buffer on ice.

1. Determine the number of CUT&Tag reactions to be run. We strongly suggest including a reaction for the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751. The negative control Normal Rabbit IgG #2729 or Normal Mouse IgG #68860 is optional, depending on the requirements of the peak calling algorithm to be used.

2. Carefully resuspend Concanavalin A Magnetic Beads into a homogeneous slurry by gently pipetting up and down, making sure not to splash any bead suspension out of the tube. 

NOTE: Avoid vortexing of the Concanavalin A Magnetic Bead suspension throughout the protocol as repeated vortexing may displace the Concanavalin A from the beads. 

3. Transfer 10 µl of the bead suspension for each CUT&Tag reaction to a new 1.5 ml microcentrifuge tube. If planning to do more than 14 CUT&Tag reactions at one time, use two or more 1.5 ml microcentrifuge tubes. No more than 140µl of Concanavalin A beads should be added to each 1.5 ml microcentrifuge tube.

4. Add 100 µl of Concanavalin A Bead Activation Buffer per 10 µl of beads. Gently mix beads by pipetting up and down.

5. Place tube on a magnetic rack for 30 sec to 2 min and then remove the supernatant using a pipette. 

NOTE: To avoid loss of beads, do NOT aspirate using a vacuum throughout the protocol.

6. Remove tubes from the magnetic rack. Wash the beads a second time by repeating steps 4 and 5.

7. Add a volume of Concanavalin A Bead Activation Buffer equal to the initial volume of bead suspension added (10 µl per reaction) and resuspend by pipetting up and down. 

NOTE: The activated beads can be stored on ice for up to 8 hrs.

II. Cell and Tissue Preparation

For most cell types, live cells can be used in the CUT&Tag assay to generate robust enrichment of histones, transcription factors, and cofactors. We strongly recommend using live cells whenever possible. For certain cell types that are fragile or sensitive to Concanavalin A, please refer to Appendix A for a light fixation protocol of cells prior to CUT&Tag. Please note that cell fixation does not increase CUT&Tag signals and over-fixation can be detrimental to the tagmentation reaction. 

Fresh tissues can also be used in the CUT&Tag assay to generate robust enrichment of histones. However, non-histone targets such as transcription factors and cofactors are not well enriched in the CUT&Tag assay. For analysis of transcription factors and cofactors in tissues, we recommend using the CUT&RUN Assay Kit #86652. Fresh tissues typically generate comparable or stronger CUT&Tag signals than fixed tissues. If fixation is necessary, refer to Appendix B for a light fixation protocol of tissues prior to the CUT&Tag experiment.

Our CUT&Tag assay works with a variety of different cell and tissue types and a wide range of starting material amounts. We recommend using 100,000 cells or 1 mg of tissue per reaction. If starting cell number is limited, histone modification targets may work with as few as 5,000 to 10,000 cells per reaction, and transcription factors and cofactors may work with as few as 20,000 cells per reaction. Success of low input reactions depends on target abundance and antibody sensitivity. An adequate amount of starting material is critical for desired CUT&Tag signal, especially for transcription factors and cofactors. Up to 250,000 cells or 5 mg tissue can be used per reaction. Buffer volumes throughout the protocol used in one reaction do not need to be adjusted based on the cell number or tissue mass, as long as the values fall within the designated range (5,000-250,000 cells or 1-5 mg of tissue). When indicated, buffer volumes do need to be scaled proportionally to the number of reactions being performed. 

The amount of digitonin recommended for cell permeabilization is in excess and should be sufficient for permeabilization of most cell lines and tissue types. However, not all cell lines and tissues exhibit the same sensitivity to digitonin. If your specific cell line or tissue does not work with the recommended digitonin concentration, you can optimize conditions by following the protocol provided in Appendix C. Digitonin treatment should result in permeabilization of > 90% of the cell population.

A. Live Cell Preparation

Before Starting:

! All buffer volumes should be scaled proportionally to the number of CUT&Tag reactions being performed.

• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287 before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.

• Prepare Complete Wash Buffer (2 ml for each cell preparation and an additional 100 µl for each CUT&Tag reaction) and keep at room temperature. For example, if using both untreated and drug-treated cells (2 cell preparations) and testing with 4 antibodies (positive control H3K4me3 #9751, negative control IgG #2729 or #68860, and two experimental antibodies; 8 reactions), a total of 4.8 ml of Complete Wash Buffer will be needed.

NOTE: All steps for live cell preparation should be performed in succession at room temperature to minimize stress on the cells. Do not vortex cell samples to avoid DNA fragmentation and cavitation of cells. 

1. Harvest 100,000 live cells for each reaction at room temperature to minimize stress on the cells. 

NOTE:  For adherent cells, detach them from the dish using Trypsin and neutralize with at least 3 volumes of tissue culture medium. We do not recommend scraping the cells from the dish to prevent cell lysis. Cells should be counted accurately using a hemocytometer or a cell counter to ensure that an accurate number of cells are being used for the experiment.  

2. Centrifuge cell suspension for 3 min at 600 x g at room temperature and remove the supernatant.

NOTE: If working with fewer than 100,000 total cells and the centrifuged cell pellet is not visible by eye, we recommend skipping the wash steps 3 to 5 below and moving directly to Step 6. After the initial centrifugation of the cell suspension in Step 2, remove most of the supernatant, leaving behind ≤40 µl of supernatant per reaction. Then in Step 6, add enough Complete Wash Buffer to the cell suspension to achieve a total volume of 100 µl per reaction.

3. Resuspend cell pellet in 1 ml of Complete Wash Buffer at room temperature by gently pipetting up and down.

4. Centrifuge for 3 min at 600 x g at room temperature and remove the supernatant.

5. Wash the cell pellet a second time by repeating steps 3 and 4 one time.

6. Add 100 µl of Complete Wash Buffer per reaction and resuspend the cell pellet by gently pipetting up and down.

7. Immediately proceed to Section III.

B. Fresh Tissue Sample Preparation

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&Tag reactions being performed.

• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287  before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.

• Prepare Complete Wash Buffer (3 ml for each tissue type and additional 100 µl for each reaction) and keep it at room temperature to minimize stress on the cells. For example, if using wild type and transgenic liver as starting material (2 tissue types) and testing 4 antibodies (positive control H3K4me3 #9751, negative control IgG #2729 or #68860, and two experimental antibodies; 8 reactions), a total of 6.8 mL of Complete Wash Buffer is needed.

1. Weigh 1 mg fresh tissue for each reaction. 

2. Place tissue sample in a dish and finely mince using a clean scalpel or razor blade. Keep dish on ice. It is important to keep the tissue cold to avoid protein degradation.

3. Resuspend tissue in 1 ml of Complete Wash Buffer and transfer the sample to a Dounce homogenizer. 

4. Disaggregate tissue pieces into a single-cell suspension with 20-25 strokes until no tissue chunks are observed.

5. Transfer cell suspension to a 1.5 ml tube and centrifuge at 3,000 x g for 3 min at room temperature, and pipette to remove supernatant from cells.

6. Resuspend the cell pellet in 1 ml of Complete Wash Buffer.

7. Centrifuge cell suspension for 3 min at 3,000 x g at room temperature and remove the supernatant.

8. Wash the cell pellet a second time by repeating steps 6 and 7 one time.

9. Add 100 µl of Complete Wash Buffer per reaction and resuspend the cell pellet by gently pipetting up and down.

10. Immediately proceed to Section III.

III. Binding of Concanavalin A Beads and Primary Antibody

NOTE: For all incubation steps in Sections III-V, it is not necessary to mix samples by rocking or rotation. Instead, we recommend simply placing sample tubes in a rack at the designated temperatures. Mixing the samples during the incubation steps does not increase the performance of the assay. Instead, rotation or rocking the samples may lead to increased bead clumping and bead loss due to potential sticking on the tube walls and caps. 

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&Tag reactions being performed.

• Warm Digitonin Solution #16359 at 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution #16359 on ice during use. Store at -20°C when finished for the day.

• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287  before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.

• Prepare 100 µl of Complete Antibody Binding Buffer per reaction and place on ice.

1. Thoroughly mix the activated Concanavalin A Beads prepared in Section I Step 7 by gently pipetting up and down. Add the beads suspension to the washed cell suspension prepared in Section II-A Step 6, or Section II-B Step 9.

2. Incubate the sample for 5 min at room temperature.

3. Place the tube on the magnetic rack for 30 sec to 2 min, then remove and discard the supernatant.

4. Remove tube from the magnet. Add 100 µl of Complete Antibody Binding Buffer per reaction and mix gently by pipetting.

5. Aliquot 100 µl of the cell:bead suspension into separate 1.5 ml tubes for each reaction and place on ice.

6. Add the appropriate amount of primary antibody to each reaction and mix gently by pipetting up and down.

NOTE: The amount of antibody required for CUT&Tag varies and should be determined by the user. For the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, or the negative control Normal Rabbit IgG #2729 or Normal Mouse IgG #68860, add 2 µl of antibody to the reaction. If possible, we highly recommend using CUT&Tag-validated antibodies in the assay. CST offers a selection of CUT&Tag validated antibodies with supporting data and appropriate dilution ratios.

7. Incubate samples at room temperature for 1 hour. This step can be extended to overnight at 4°C. 

8. Immediately proceed to Section IV.

IV. Binding of Secondary Antibody

Before Starting:

! All buffer volumes should be scaled proportionally to the number of CUT&Tag reactions being performed.

• Warm Digitonin Solution #16359 at 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution #16359 on ice during use. Store at -20°C when finished for the day.

• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287  before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.

• Freshly prepare 1.05 ml of Digitonin Buffer per reaction and place on ice.  

NOTE: Digitonin Buffer prepared here will be used for both Section IV and V.

1. Make secondary antibody pre-mix. For each reaction, dilute 1 µl of Goat Anti-Rabbit IgG (H+L) Antibody #35401 or 1 µl of Donkey Anti-Mouse IgG (H+L) Antibody #52885 into 50 µl Digitonin Buffer. Proportionally scale up the secondary antibody pre-mix based on the number of reactions. Mix by gently pipetting up and down and place on ice.

2. Place the sample tubes containing the primary antibody incubation solution from Section III Step 7 on the magnetic rack for 30 sec to 2 min and then remove the supernatant.

3. Add 50 µl of secondary antibody pre-mix to each sample tube and gently mix the sample by pipetting up and down.

4. Incubate samples at room temperature for 30 min. 

5. Immediately proceed to Section V.

V. Binding of pAG-Tn5 Enzyme and Tagmentation

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&Tag reactions being performed.

• Make sure the 10% SDS Solution #20533 is completely in solution. Warming it up at 37°C can help to dissolve the SDS precipitates. 

• Warm Digitonin Solution #16359 at 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution #16359 on ice during use. Store at -20°C when finished for the day.

• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287  before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.

• Prepare 1.2 ml of High Salt Digitonin Buffer per reaction and place on ice.

• Prepare 150 µl of Tagmentation Buffer per reaction and place on ice.

1. For each reaction, make a pAG-Tn5 pre-mix by diluting 2 µl of CUT&Tag pAG-Tn5 (Loaded) #79561 into 50 µl of High Salt Digitonin Buffer. Proportionally scale up the pAG-Tn5 pre-mix based on the number of reactions. Mix by gently pipetting up and down and place on ice.

2. Place the sample tubes containing the secondary antibody incubation solution from Section IV Step 4 on the magnetic rack for 30 sec to 2 min and then remove the supernatant.

3. Remove tubes from the magnetic rack and add 500 µl of Digitonin Buffer prepared in Section IV. Resuspend beads by gently pipetting up and down.

4. Place the tubes on the magnetic rack for 30 sec to 2 min and then remove the supernatant.

5. Repeat steps 3 and 4 one time for a second wash.

6. Remove tubes from magnetic rack. Add 50 µl of pAG-Tn5 pre-mix to each tube and gently mix the sample by pipetting up and down.

7. Incubate samples at room temperature for 1 hr.

8. Place the tubes on the magnetic separation rack for 30 sec to 2 min and then remove the supernatant.

9. Remove tubes from the magnetic separation rack. Add 500 µl of High Salt Digitonin Buffer and resuspend beads by gently pipetting up and down.

10. Place the tubes on the magnetic rack for 30 sec to 2 min and then remove the supernatant.

11. Repeat steps 9 and 10 one time for a second wash.

12. Remove tubes from magnetic rack. Add 150 µl of Tagmentation Buffer to each tube and mix by pipetting up and down.

13. Incubate samples at 37°C for 1 hr.

14. To stop tagmentation, add 6.75 µl of 0.5 M EDTA #7011, 8.25 µl of 10% SDS #20533 and 1.5 µl of 20 mg/mL Proteinase K #10012 to each sample and mix by a quick vortex.

15. Incubate samples at 58°C for 1 hr to release tagmented chromatin fragments into solution. This incubation can be extended overnight. If incubating overnight, use safe-lock tubes to prevent sample evaporation.

NOTE: If starting with fixed cells or tissues, incubate samples at 65°C for 2 hr in safe-lock tubes to sufficiently reverse cross-links. This incubation can be extended overnight.

16. Centrifuge tubes at room temperature for 2 min at 16,000x g and place the tubes on a magnetic rack for 30 sec to 2 min.

17. Transfer the supernatants to new 1.5 ml tubes. These are your CUT&Tag DNA samples to be purified.

18. Proceed to Section VI. (SAFE STOP) Alternatively, samples can be stored at -20°C for up to 1 week. However, be sure to warm samples to room temperature before DNA purification (Section VI).

VI. DNA Purification

Before Starting:

• Please equilibrate DNA Purification Columns, DNA Binding Buffer, DNA Wash Buffer, and DNA Elution Buffer to room temperature before use.

• !! Add 24 ml of ethanol (96-100%) to DNA Wash Buffer before use. This step only has to be performed once prior to the first set of DNA purifications.

• Use one DNA Purification collection tube for each CUT&Tag DNA sample to be purified.

1. Add 833 µl DNA Binding Buffer to each CUT&Tag DNA sample and mix by pipetting up and down.

NOTE: 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.

2. Transfer 600 µl of each sample from Step 1 to a DNA spin column in collection tube.

3. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.

4. Remove the spin column from the collection tube and discard the liquid. Replace the spin column in the empty collection tube.

5. Repeat steps 2-4 until the entire sample from Step 1 has been spun through the spin column. Replace the spin column in the empty collection tube.

6. Add 750 µl of DNA Wash Buffer to the spin column in collection tube.

7. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.

8. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the empty collection tube.

9. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.

10. Discard collection tube and liquid. Retain spin column.

11. Add 30 µl of DNA Elution Buffer to each spin column and place into a clean 1.5 ml tube. 

NOTE: To fully elute DNA from the columns, a minimum volume of 30 µl of DNA Elution Buffer is required.

12. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec to elute DNA.

13. Remove and discard DNA spin column. Eluate is now purified DNA. (SAFE STOP) Samples can be stored at -20°C for up to 6 months.

NOTE: Considering the typical low yield of CUT&Tag DNA, we strongly recommend using all of the 30 µl of DNA sample for library amplification.

VII. NG-Sequencing Library Construction

The immuno-enriched DNA samples prepared with this kit are directly compatible with NG-seq. For downstream NG-seq DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina Systems platforms, we recommend using the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415. Please note that the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 and Multiplex Oligos for Illumina Systems (ChIP-seq, CUT&RUN) #29580 or #47538 are not compatible with CUT&Tag DNA samples.

Additional Recommendations for DNA Library Preparation:

• The yield of the amplified CUT&Tag DNA library can vary based on the DNA quantification method used. If using the Nanodrop or QIAxpert Systems, the expected reading is 10-20 ng/µl for histone targets and 5-12 ng/µl for non-histone targets. If the library concentration is lower than 3 ng/µl with the Nanodrop or QIAxpert Systems, please refer to the troubleshooting guide before sequencing your samples. If using the Qubit Fluorometric Quantification system or the Picogreen assay, the expected reading is 3-10 ng/µl for histone targets and could be lower than 1 ng/µl for non-histone targets. Because of these low concentrations, the Bioanalyzer or TapeStation systems may generate a profile with very weak or even no visible peaks, especially for targets that are not abundant in cells. In these cases, we still recommend continuing with NGS if the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 generates the expected library yield and/or Bioanalyzer peaks, indicative of an overall successful experiment. Please refer to our CUT&Tag FAQ web page for supporting data or if extra guidance is needed to pool together samples with a variety of yields.

• A sequencing depth of 2 million reads per sample is usually sufficient for CUT&Tag assay, regardless of target types. The duplication rate of reads significantly increases if the sequencing depth is greater than fifteen million per sample. The signal to noise ratio decreases if the sequencing depth is lower than one million reads per sample.

• If starting with less than 20,000 cells, it is common to obtain lower mapping rates or higher duplication rates in the NGS reads. If this happens, we recommend increasing the sequencing depth to obtain enough unique mapped reads for downstream data analysis. 

APPENDIX A: Fixed Cell Preparation

We strongly recommend using live cells whenever possible. For certain cell types that are fragile or sensitive to concanavalin A, please refer to the protocol below to lightly fix cells prior to the CUT&Tag experiment. Please note that cell fixation does not significantly increase CUT&Tag signals. In fact, over-fixation may lead to weaker CUT&Tag signals. Refer to the description in Section II to determine the appropriate cell number in each reaction. 

NOTE: The following reagents are required for fixed cell preparation and are not included in this kit: 37% formaldehyde or 16% Formaldehyde Methanol-Free #12606 and Glycine Solution (10X) #7005.

Before Starting:

! All buffer volumes should be scaled proportionally to the number of CUT&Tag reactions being performed.

• Prepare 2.7 µl of 37% formaldehyde or 6.25 µl of 16% Formaldehyde, Methanol-Free #12606 per 1 ml of cell suspension to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer’s expiration date.

• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287  before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.

• Prepare Complete Wash Buffer (2 ml for each cell preparation and an additional 100 µl for each CUT&Tag reaction) and keep it at room temperature. For example, if using both untreated and drug-treated cells (2 cell preparations) and testing with 4 antibodies (positive control H3K4me3 #9751, negative control IgG #2729 or #68860, and two experimental antibodies; 8 reactions), a total of 4.8 ml of Complete Wash Buffer will be needed.

1. Harvest 100,000 live cells for each reaction. 

NOTE:  For adherent cells, detach them from the dish using Trypsin and neutralize with at least 3 volumes of tissue culture medium. We do not recommend scraping the cells from the dish to prevent cell lysis. Cells should be counted accurately using a hemocytometer or other cell counter to ensure the proper number of cells are being used for the experiment.  

2. Add 2.7 µl of 37% formaldehyde or 6.25 µl of 16% Formaldehyde, Methanol-Free #12606 per 1 ml of cell suspension to achieve a final concentration of 0.1% formaldehyde. Swirl tube to mix and incubate at room temperature for 2 min.

3. Stop cross-linking by adding 100 µl of Glycine Solution (10X) #7005 per 1 ml of fixed cell suspension. Swirl the tube to mix and incubate at room temperature for 5 min. 

4. Centrifuge cell suspension for 3 min at 3,000 x g at 4°C and remove the supernatant. Immediately proceed to Step 5. (SAFE STOP) Alternatively, fixed cell pellets may be stored at -80°C before using for up to 6 months. 

NOTE: If working with fewer than 100,000 total cells and the centrifuged cell pellet is not visible by eye, we do NOT recommend freezing down cell pellets. Instead, we recommend continuing on with the protocol and skipping the wash steps 5 to 7 below. After the initial centrifugation of the cell suspension in Step 4, remove most of the supernatant, leaving behind ≤40 µl cell medium per reaction. Then in Step 8 add enough Complete Wash Buffer to the cell suspension to achieve a total volume of 100 µl per reaction.

5. Resuspend cell pellet in 1 ml of Complete Wash Buffer by gently pipetting up and down.

6. Centrifuge for 3 min at 3,000 x g at 4°C and remove the supernatant.

7. Wash the cell pellet a second time by repeating steps 5 and 6 one time. 

8. For each reaction, add 100 µl of Complete Wash Buffer and resuspend the cell pellet by gently pipetting up and down.

9. Immediately proceed to Section III.

APPENDIX B: Fixed Tissue Sample Preparation

For most tissue types, 1 mg of fresh tissue is sufficient to generate robust enrichment of histones. If fresh tissue is not accessible, lightly fixed tissue (0.1% formaldehyde for 2 min) can be used. Fixed tissue samples can be frozen at -80°C up to 6 months before using. Over-fixation may lead to weaker CUT&Tag signals. The CUT&Tag assay does not work well for enrichment of transcription factors and cofactors from tissues. For analysis of transcription factors and cofactors, we recommend using the CUT&RUN Assay Kit #86652. 

NOTE: The following reagents are required for fixed tissue preparation and are not included in this kit: 37% formaldehyde or 16% Formaldehyde Methanol-Free #12606, Phosphate Buffered Saline (PBS) #9872, and Glycine Solution (10X) #7005.

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&Tag reactions being performed.

• Prepare 2.7 µl of 37% formaldehyde or 6.25 µl of 16% Formaldehyde, Methanol-Free #12606 per 1 ml of cell suspension to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

• Prepare 100 µl of Glycine Solution (10X) #7005 per 1 ml of fixation buffer.

• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287  before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.

• Prepare Complete Wash Buffer (3 ml for each tissue type and additional 100 µl for each reaction) and keep it at room temperature to minimize stress on the cells. 

• Prepare 1 ml Fixation Buffer for each tissue type. Use fresh formaldehyde that is not past the manufacturer’s expiration date.

• Prepare 1 ml of Fixation Wash Buffer for each tissue type and place on ice.

1. Weigh 1 mg fresh tissues for each reaction. 

2. Place tissue sample in a dish and finely mince using a clean scalpel or razor blade. Keep dish on ice. It is important to keep the tissue cold to avoid protein degradation.

3. Immediately transfer minced tissue to 1 ml of Fixation Buffer and swirl tube to mix.  

NOTE: This volume of fixation solution is sufficient for up to 50 mg of tissue. If processing >50 mg, scale up the amount of Fixation Buffer used in Step 3 and Fixation Wash Buffer used in Step 7 accordingly.

4. Incubate at room temperature for 2 min.

5. Stop cross-linking by adding 100 µl of Glycine Solution (10X) #7005 per 1 ml of Fixation Buffer. Swirl the tube to mix and incubate at room temperature for 5 min. 

6. Centrifuge tissue for 5 min at 2,000 x g at 4°C and remove the supernatant.

7. Resuspend tissue with 1 ml of Fixation Wash Buffer.

8. Centrifuge for 5 min at 2,000 x g at 4°C and remove the supernatant and proceed to step 9. (SAFE STOP) Alternatively, fixed tissue pellets may be stored at -80°C before disaggregation for up to 6 months.

9. Resuspend tissue in 1 ml of Complete Wash Buffer and transfer the sample to a Dounce homogenizer. 

10. Disaggregate tissue pieces into single-cell suspension with 20-25 strokes until no tissue chunks are observed.

11. Transfer cell suspension to a 1.5 ml tube and centrifuge at 3,000 x g for 3 min at room temperature, remove supernatant from cells.

12. Resuspend cell pellet in 1 ml of Complete Wash Buffer.

13. Centrifuge cell suspension for 3 min at 3,000 x g at room temperature and remove the supernatant.

14. Wash the cell pellet a second time by repeating steps 12 and 13 one time.

15. For each reaction, add 100 µl of Complete Wash Buffer and resuspend the cell pellet by gently pipetting up and down.

16. Immediately proceed to Section III.

APPENDIX C: Determination of Cell Sensitivity to Digitonin

In the CUT&Tag protocol, the addition of digitonin to the buffers facilitates the permeabilization of cell membranes and entry of the primary antibody, secondary antibody, and pAG-Tn5 enzyme into the cells and nuclei. Therefore, having an adequate amount of digitonin in the buffers is critical to the success of antibody and enzyme binding, and digestion of targeted genomic loci. Different cell lines exhibit varying sensitivities to digitonin cell permeabilization. While the amount of digitonin recommended in this protocol should be sufficient for permeabilization of most cell lines or tissues, you can test your specific cell line or tissue using this protocol. We have found that the addition of excess digitonin is not deleterious to the assay, so there is no need to perform a concentration curve. Rather, a quick test to determine if the recommended amount of digitonin works for your cell line is sufficient.

Before Starting:

• Warm Digitonin Solution #16359 at 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution #16359 on ice during use. Store at -20°C when finished for the day.

• Prepare 100 µl of 1X Wash Buffer per reaction. It is not necessary to add spermidine or Protease Inhibitors in the buffer for this test.

1. In a 1.5 ml tube, collect 100,000 cells (from Section II-A, Step 1), centrifuge for 3 min at 600 x g at room temperature and withdraw the supernatant. For tissue, collect disaggregated cells from 1 mg of tissue (from Section II-B, Steps 1-8). 

2. Resuspend cell pellet in 100 µl of 1X Wash Buffer.

3. Add 2.5 µl Digitonin Solution #16359 to each reaction and incubate for 10 min at room temperature.

4. Mix 10 µl of cell suspension with 10 µl of 0.4% Trypan blue stain.

5. Use a hemocytometer or cell counter to count the number of stained cells and the total number of cells. Sufficient permeabilization results in > 90% of cells staining with Trypan blue.

6. If less than 90% of cells stain with Trypan blue, then increase the amount of Digitonin Solution #16359 added to each reaction and repeat steps 1-5 until > 90% cells are permeabilized and stained. Use this amount of Digitonin Solution #16359 in Sections I - V.

APPENDIX D: Troubleshooting Guide