This article describes the histone western blot protocol, which is often used at Abcam for identifying histone proteins obtained from purified calf thymus.
- For each lane, either 0.5 μg of calf thymus or acid extracted histones diluted in 1X LDS sample buffer supplemented with 100 mM DTT is prepared. The sample is heated for 5 minutes at 95 °C and then briefly centrifuged to restore its volume from condensation created in the tube.
It must be noted that protein loadings obtained from cellular lysates will have to be empirically established.
- A 10% Bis-Tris gel of 1.0 mm thickness is prepared. To obtain a clear and more effective resolution of histone proteins, a higher percentage gel (15%) is proposed.
- The histone samples are loaded, making sure that a pre-stained protein standard is also included. The gel is then run in MES SDS running buffer for 35 minutes at 200 V.
When handling smaller protein resolutions, it would be helpful if the dye front does not run completely off the gel.
- When transferring proteins from the gel, users can refer to the protocols provided with their transfer apparatus, as these will differ based on the method of transfer used – i.e. wet blotting or semi-dry blotting. For effective protein transfer, 30 to 90 minutes transfer times should be adequate. Here, 1X transfer buffer / 20% methanol was used to transfer at 30 V for 70 minutes.
For optimal retention of histone proteins, it is advisable to use nitrocellulose membranes with a pore size of 0.2 mm.
- Ponceau staining is performed to validate the effective transfer and equal loading of the histone proteins. dH2O is then added to dilute the Ponceau from the membrane.
- Using 5% BSA / 0.1% TBST (50 mM Tris-HCl, pH 7.5, 150mM NaCl, 0.1% Tween 20), the membrane is blocked at room temperature (RT) for 1 hour.
- If required, the membrane is cut into strips and the primary antibody is prepared by diluting in blocking buffer (5% BSA / 0.1% TBST) at a dilution proposed by the Abcam datasheet. Blocking peptides (at 1 μg/ml) are then added as required and incubated on a rotating platform at RT for 20 minutes. The membrane is incubated with the primary antibody at RT or overnight for 1.5 hours at 4 °C.
- The blots are briefly washed in 0.1% TBST, and then washed twice for 5 minutes and again twice for 10 minutes using the same buffer
- The membrane is incubated with the secondary antibody for 1 hour at RT [for instance ab6721 –Goat polyclonal to rabbit IgG H&L (HRP)] once again diluted as proposed in 1% BSA / 0.1% TBST.
- The membrane is washed twice in 0.1% TBST for 5 minutes, and again twice for 10 minutes.
- ECF, ECL, or infrared detection is carried out as recommended by the manufacturer, for example, ECL reagents are added at RT for 3 minutes and WB image is captured using different durations of exposure: 10 seconds, 30 seconds, 1 minutes, 2 minutes, 3 minutes, 4 minutes, and 5 minutes.
Top tips for successful western blotting with Abcam’s range of histone antibodies
- A high percentage gel should be used to achieve a clear resolution of histone proteins
- Nitrocellulose membrane with a pore size of 0.2 µm should be used for optimal retention of histone proteins
- A high-quality BSA should be used in the blocking solutions instead of the standard dried milk such as Marvel
- Loading control antibodies should always be used to standardize the experiments
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