Western Blot protocol specific for SR-BII Antibody (NB400-102)

Procedure Guide for NB 400-102 Polyclonal anti-SR-B11

Western Blot Procedure

1. Run ~50 ug of protein on a 4-20% Tris-glycine mini-gel at 125V for 90 minutes.
2. Equilibrate gel, nitrocellulose membrane, Whatman paper, and blotting pads in transfer buffer for 15 minutes.
3. Transfer protein to the membrane at 25V for 90 minutes.
4. Allow membrane to air-dry.
5. Block membrane with blocking buffer [1XTBS / 5% NFDM / 0.1% Tween-20] for 1 hour at room temperature (~23-27 degrees C).
6. Wash membrane twice, for 5 minutes each, with 1XTBS.
7. Incubate membrane with 1:1,000 dilution of NB400-102 (anti-SR-BII), diluted in blocking buffer, overnight at 4C.
8. Wash membrane once for 15 minutes, then four times for 5 minutes each, with TBST.
9. Incubate membrane with 1:10,000 dilution of goat anti-rabbit IgG-HRP (BioRad), diluted in blocking buffer, for 35 minutes at room temperature.
10. Wash membrane once for 15 minutes, then four times for 5 minutes each, with TBST.
11. Detect cross-reacting proteins using ChemiGlow reagents from Alpha Innotech.

NOTE: Jurkat whole cell extracts (NB800-PC2) and HL-60 whole cell extracts (NB800-PC3) were used as a positive control for this antibody.