Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

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

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). 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.

IP

Sample preparation

1. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
2. Remove PBS and add ice-cold 1X cell lysis buffer to each plate and incubate on the ice for 5 min.
3. Scrape cells off the plate and transfer to themicrocentrifuge tubes. Keep on the ice.
4. Sonicate on the ice three times for 5 sec each.
5. Centrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. If necessary, store itin -80°C.

Cell Lysate Pre-Clearing (Optional)

Pre-clearing the lysate can help reduce non-specific binding and reduce background.

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

Immunoprecipitation

1. Add primary antibody at the appropriate dilutionto cell lysat. Incubate with gentle agitation or rotation overnight at 4°C.
2. Add protein Aagarose(10–30 µl of 50% bead slurry). Incubate with gentle agitation or rotation for 1–3 hr at 4°C.
3. Centrifuge for 30 sec at 4°C and discard the supernatant. Wash the pellet five times with 500 µl of 1X cell lysis buffer. Keep on the ice.
4. Continue with sample analysis.

Sample Analysis

a. Western Immunoblotting

1. Resuspend the pellet with SDS sample buffer. Vortex, then centrifuge for 30 sec at 14,000 x g.
2. Heat the samples to 95–100°C for 2-5 min and centrifuge for 1 min at 14,000 x g.
3. Load the appropriate amount ofsamples on a 4–20% gel for SDS-PAGE.
4. Analyze sample by western blot.

b. Kinase Assay

1. Wash the pellet twice with 500 µl 1X kinase buffer. Keep on the 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 the reaction by adding SDS sample buffer. Vortex to mix, then centrifuge for 30 sec.
5. Transfer supernatantcontaining phosphorylated substrate to another tube.
6. Heat the sample to 95–100°C for 2–5 min and centrifuge for 1 min at 14,000 x g.Load the sample on SDS-PAGE.

Immunohistochemistry (Paraffin)

Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:

1. Incubate sections in three washes of xylene for 5 min each.
2. Incubate sections in two washes of 100% ethanol for 10 min each.
3. Incubate sections in two washes of 95% ethanol for 10 min each.

2. Wash sections two times in dH2O for 5 min each.

Antigen retrieval

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections:
1. Incubate sections in 95% ethanol two times for 10 sec each.
2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
3. Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.

Immunofluorescence (Immunocytochemistry)

Specimen Preparation (forcultured cell lines, IF-IC)

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. Fix cells for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining.

Immunostaining

1. Add theblocking buffer and incubate for 60 min at RT.
2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
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 specimens 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. Mount slides usingmounting medium with DAPI and cover with coverslips.
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

Fixation

NOTE: lyse red blood cells and wash by centrifugation prior to fixation if using whole blood.
1. Collect cells by centrifugation and discardthe supernatant.
2. Resuspendthe cells in 0.5-1 ml 1X PBS and Add formaldehyde to obtain thefinal concentration of 4%.
3. Fix for 15 min at room temperature.
4. Wash by centrifugation with excess 1X PBS. Discard the supernatant and resuspendthe cells in 0.5-1 ml 1X PBS.

Permeabilization

1. Permeabilize the cells by adding ice-cold 100% methanol slowly, with gently vortexing.The final concentration of methanol is 90%.
2. Incubate the cells on the ice for 30 min.
3. Proceed with immunostaining or store the cells at -20°C in 90% methanol.

Immunostaining

NOTE: Count the cells using a hemocytometer or an alternative method.
1. Aliquot a desired number of cells into tubes or wells.
2. Wash the cellsin excess 1X PBS by centrifugation to remove methanol. Discard the supernatant. Repeat it if necessary.
3. Resuspendthe cells in 100 µl of diluted primary antibody prepared in incubation buffer.
4. Incubate for 1 hr at room temperature.
5. Wash the cells in incubation bufferand separated by centrifugation. Discard the supernatant. Repeat it twice.
6. Resuspendthe cells in 100 µl of diluted fluorochrome-conjugated secondary antibody.
7. Incubate for 30 min at room temperature.
8. Wash the cells in incubation buffer and removed the supernatant by centrifugation. Repeat it twice.
9. Resuspendthe cells in 1X PBS and analyze using flow cytometer (If the sample is also used for DNA staining, proceed to the next part).

E. Optional DNA Dye

1. Resuspendthe cells in DNA dye.
2. Incubate for at least 5 min at room temperature.
3. Analyze cells in DNA staining solution using flow cytometer.

Chromatin IP

Cell Culture Cross-linking and Sample Preparation

For optimal ChIP results, use approximately 4 X 106 cells for each experiment. One additional sample should be processed for Analysis of Chromatin Digestion and Concentration. Include one extra dish of cells in experiment to be used for determination of cell number using a hemocytometer.
• Before starting

• Warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawedprior to use.
• Prepare 2 ml of PBS + 10 µl 200X PIC per 15 cm dish and place it onice.
• Prepare 40 ml of PBS per 15 cm dish to be processed and place it onice.
• Prepare 540 µl of 37%(w/v) formaldehyde per 15 cm dish of cells and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

1. To crosslink proteins to DNA, add 540 µl of 37%(w/v) formaldehyde to each 15 cm culture dish containing 20 ml medium. Swirl briefly to mix and incubate 10 min at room temperature. The final concentration of formaldehyde is 1%.
2. To quench formaldehyde, add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min at room temperature.
3. For suspension cells, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm for 5 min at 4°C and wash pellet two times with 20 ml ice-cold PBS. Remove the supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion.
4. For adherent cells, discardthe media and wash the cellstwice with 20 ml ice-cold 1X PBS.
5. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape the cells into cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
6. Centrifuge the cells at 1,500 rpm for 5 min at 4°C. Remove the supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion.

Nuclei Preparation and Chromatin Digestion

Before starting
• Warm 200X Protease Inhibitor Cocktail (PIC). Make sure it is completely thawed prior to use.
• Prepare 1 M DTT. Make sure DTT crystals are completely in solution.
IMPORTANT: Once in solution, store 1M DTT at -20°C.
• Warm 10X ChIP Buffer and ensure SDS is completely in solution.
• Prepare 1 ml 1X Buffer A + 0.5 µl 1M DTT + 5 µl 200X PIC per IP prep and place it on ice.
• Prepare 1.1 ml 1X Buffer B+ 0.55 µl 1M DTT per IP prep and place it on ice.
• Prepare 100 µl 1X ChIP Buffer + 0.5 µl 200X PIC per IP prep and place it on ice.

IMPORTANT: Steps in this part must be performed on ice or at 4 ℃
1. Resuspendthe cells in 1 ml ice-cold 1X Buffer A + DTT + PIC. Incubate onice for 10 min. Mix by inverting tube several times.
2. Centrifuge at 3,000 rpm for 5 min at 4°C to pellet nuclei. Remove the supernatant and resuspendthe pellet in 1 ml ice-cold 1X Buffer B + DTT. Repeat the step one more time, thenresuspendthe pellet in 100 µl 1X Buffer B +DTT. Transfer to a 1.5 ml microcentrifuge tube (up to 1 ml total per tube).
3. For chromationdigestion, add Micrococcal Nuclease into the tube, mix by inverting tube several times and incubate for 20 min at 37°C with frequent shaking to digest DNA to length of approximately 150-900 bp.Mix by inversion every 3-5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and cell lines.
4. Stop digestion by adding 10 µl of 0.5 M EDTA per IP prep.
5. Centrifuge at 13,000 rpm for 1 min at 4°C to pellet nuclei and remove supernatant.
6. Resuspendthe nuclear pellet in 100 µl of 1X ChIP Buffer + PIC and incubate on ice for 10 min.
7. Sonicate up to 500 µl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. Alternatively, nuclei can be lysed using ahomogenizer.
8. Clarify lysates by centrifugation at 10,000 rpm for 10 min at 4°C.
9. Transfer supernatant to a new tube. The product is the cross-linked chromatin preparation. Stored at -80°C for further use. (Remove 50 µl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration)

Analysis of Chromatin Digestion and Concentration

1. To the 50 µl chromatin sample, add 100 µl nuclease-free water, 6 µl 5 M NaCl, and 2 µl RNAse A. Vortex to mix and incubate at 37°C for 30 min.
2. Add 2 µl Proteinase K. Vortex to mix and incubate at 65°C for 2 h.
3. Purify DNA from samples using DNA purification spin columns.
4. After purification of DNA, remove a 10 µl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be digested to a length of approximately 150-900 bp (1 to 5 nucleosomes).
5. Determine the concentration of DNA.
NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion of chromatin may diminish signal in the PCR quantification. Under-digestion of chromatin may lead to increased background signal and lower resolution. Adding too little chromatin to the IP may result in diminished signal in the PCR quantification.

Chromatin Immunoprecipitation

For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin per IP reaction. Typically, 100 µl of digested chromatin is diluted into 400 µl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 µl of chromatin is required per IP, the cross-linked chromatin preparation does not need to be diluted as described below. Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes.

Before starting
• Warm 200X Protease Inhibitor Cocktail (PIC). Make sure PIC is completely thawed.
• Warm 10X ChIP Buffer and ensure SDS is completely in solution.
• Thaw digested chromatin preparation and place on ice.
• Prepare low salt wash: 3 ml 1X ChIP Buffer (300 µl 10X ChIP Buffer + 2.7 ml water) per reaction. Store at room temperature until use.
• Prepare high salt wash: 1 ml 1X ChIP Buffer (100 µl 10X ChIP Buffer + 900 µl water) + 70 µl 5M NaCl per reaction. Store at room temperature until use.

1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into the desired number of reactions: 400 µl of 1X ChIP Buffer (40 µl of 10X ChIP Buffer + 360 µl water) + 2 µl 200X PIC per reaction. When determining the number of reactions, remember to include the positive control and negative control samples. Place the mix on ice.
2. To the prepared 1X ChIP Buffer, add the equivalent of 100 µl (5 to 10 µg of chromatin) of the digested, cross-linked chromatin preparation per reaction.
3. Remove a 10 µl sample of the diluted chromatin and transfer to a microcentrifuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use.
4. For each immunoprecipitation reaction, transfer 500 µl of the diluted chromatin to a 1.5 ml microcentrifuge tube and add the antibody. The amount of antibody required per IP varies and should be determined by the user. Incubate IP samples 4 h to overnight at 4°C with rotation.
5. Resuspend Protein A/G Magnetic Beads by gently vortexing. Immediately add 30 µl of Protein A/G Magnetic Beads to each IP reaction and incubate for 2 h at 4°C with rotation.
6. Pellet protein A/G magnetic beads by placing the tubes in a magnetic separator. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.
7. Wash magnetic beads by adding 1 ml of low salt wash and incubate at 4°C for 5 min with rotation. Repeat steps 6 and 7 two additional times.
8. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
9. Pellet magnetic beads by placing the tubes in a Magnetic Separator. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.

Elution of Chromatin and Reversal of Cross-links

Before starting
• Warm 2X ChIP Elution Buffer in a 37°C water bath and ensure SDS is in solution.
• Set a water bath or thermomixer to 65°C.
• Prepare 150 µl 1X ChIP Elution Buffer for each reaction and 2% input sample.

1. Add 150 µl of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at room temperature until Step 6.
2. Add 150 µl 1X ChIP Elution Buffer to each IP sample.
3. Elute chromatin from the antibody/magnetic beads for 30 min at 65°C with gentle vortexing (1,200 rpm).
4. Pellet protein A/ G magnetic beads by magnetic separation.
5. Carefully transfer eluted chromatin supernatant to a new tube.
6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 µl 5M NaCl and 2 µl Proteinase K, and incubate 2 h at 65°C. This incubation can be extended overnight.
7. Immediately proceed to the next part. Alternatively, samples can be stored at -20°C. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Buffer.

DNA Purification

This part was performed at room temperature
1. Add 750 µl DNA Binding Buffer to each DNA sample and vortex briefly.
o 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
2. Transfer 450 µl of each sample from Step 1 to a DNA spin column in collection tube.
3. Centrifuge at 14,000 rpm for 30 sec.
4. Discard the liquid in the collection tube andreplace spin column in the collection tube.
5. Transfer the remaining 450 µl of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
6. Add 750 µl of DNA Wash Buffer (ethanol added) to the spin column in collection tube.
7. Centrifuge at 14,000 rpm for 30 sec.
8. Discard the liquid in the collection tube andreplace spin column in the collection tube.
9. Centrifuge at 14,000 rpm for 30 sec.
10. Discard collection tube and liquid. Retain spin column.
11. Place the spin column in a new, clean 1.5 ml microcentrifuge tube and add 50 µl of DNA Elution Buffer to each spin column.
12. Centrifuge at 14,000 rpm for 30 sec to elute DNA.
13. Remove and discard DNA spin column. Eluent in the microcentrifuge tube is purified DNA. Samples can be stored at -20°C.

Data analysis

Purified DNA can be used in PCR (standard PCR or Real-Time Quantitative PCR).