|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
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
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.
1. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
Cell Lysate Pre-Clearing (Optional)
Pre-clearing the lysate can help reduce non-specific binding and reduce background.
1. Vortex to mix beads.
1. Add primary antibody at the appropriate dilutionto cell lysat. Incubate with gentle agitation or rotation overnight at 4°C.
a. Western Immunoblotting
1. Resuspend the pellet with SDS sample buffer. Vortex, then centrifuge for 30 sec at 14,000 x g.
b. Kinase Assay
1. Wash the pellet twice with 500 µl 1X kinase buffer. Keep on the ice.
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. Wash sections two times in dH2O for 5 min each.
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.
1. Wash sections in dH2O three times for 5 min each.
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.
1. Add theblocking buffer and incubate for 60 min at RT.
NOTE: lyse red blood cells and wash by centrifugation prior to fixation if using whole blood.
1. Permeabilize the cells by adding ice-cold 100% methanol slowly, with gently vortexing.The final concentration of methanol is 90%.
NOTE: Count the cells using a hemocytometer or an alternative method.
E. Optional DNA Dye
1. Resuspendthe cells in DNA dye.
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.
Nuclei Preparation and Chromatin Digestion
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.
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.
Elution of Chromatin and Reversal of Cross-links
This part was performed at room temperature
Purified DNA can be used in PCR (standard PCR or Real-Time Quantitative PCR).
|Specificity||anti-Flag-tag Mouse Recombinant mAb detects the level of Flag-tagged target protein.|