实验概要

Western blot detection of histone proteins.

实验步骤

The  following protocol refers to the western blot detection of histone  proteins derived from purified calf thymus performed at Abcam.

1.        For  each lane prepare 0.5 μg calf thymus or acid extracted histones diluted  in 1X NuPAGE LDS sample buffer (Invitrogen) supplemented with 100 mM  DTT. Heat the sample to 95°C for 5 minutes. Centrifuge the sample  briefly to restore sample volume from condensation formed in the tube  during heating.

2.        Prepare  a 10% NuPAGE Bis Tris gel 1.0 mm. A higher percentage gel (15%) is  recommended for more effective resolution of histone proteins.

3.        Load  the histone samples, remembering to include a pre-stained protein  standard (Precision Plus Protein Standard (Kaleidoscope), Bio-Rad). Run  the gel in NuPAGE MES SDS running buffer at 200 V for 35 mins.
NB It is advisable not to run the dye front completely off the gel.

4.        Transfer  the protein samples onto a nitrocellulose membrane with reduced pore  sizes (Invitrogen; LC2000) at 30 V for 70 minutes using NuPAGE transfer  buffer (1X) / 20% methanol.

5.        Verify  the successful transfer and equal loading of the histones using Ponceau  staining. Dilute the Ponceau out of the membrane by adding dH2O.

6.        Block  the membrane for 1 hour at room temperature (RT) using 5% BSA / 0.1%  TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20).

7.        Cut  the membrane into strips if necessary and prepare the primary antibody  by diluting in blocking buffer (5% BSA / 0.1% TBST) at a dilution  recommended by the Abcam datasheet. Add blocking peptides  as required and incubate on a rotating platform for 20 minutes at RT.  Incubate the membrane with the primary antibody for 1.5 hours at RT or  overnight at 4°C.

8.        Rinse  the blots briefly in 0.1% TBST and then perform two 5 minute washes  followed by two 10 minute washes using the same buffer.

9.        Incubate the membrane with the secondary antibody for 1 hour at RT, diluted in 5% BSA / 0.1% TBST. (For example ab6721: Goat polyclonal to rabbit IgG H&L (HRP)).

10.    Wash the membrane in 0.1% TBST twice for 5 minutes, and twice for 10 minutes.

11.    Add  ECL reagents for 3 minutes at RT. Capture WB image using Syngene  GeneGnome using various durations of exposure: 10 s, 30 s, 1 min, 2 min,  3 min, 4 min and 5 min.

注意事项

Top tips for successful western blotting with our range of histone antibodies.

Use a high percentage gel for clear resolution of histone proteins.
Use a nitrocellulose membrane with a pore size of 0.2 μm to ensure optimal capture of histone proteins.
Use high quality BSA in your blocking solutions rather than conventional dried milk such as Marvel.