User:Vijay/Protocol/western-msa

Overview
MSA is a putative membrane protein. MSA antibody was made by ProSci INC., We used peptide cocktail approach (2 antigenic peptides) for raising antibodies. 2 Rabbits for immunization. ELISA done. Antisera obtained.

Materials

 * Perfect Western (GenHunter Corp)
 * PVDF membrane (BIORAD)
 * Wash Buffer (as per our lab western blot protocol)
 * Blocking agent (5% non-fat dry milk in Wash Buffer)
 * Primary antibody (anti-MSA)
 * Secondary antibody (Goat anti-rabbit IgG-HRP conjugate from Jackson ImmunoResearch)
 * Detection reagent (ECL from GE)

Procedure

 * 1) SDS-PAGE and TRANSFER done as per our lab protocol
 * 2) Incubate blot with blocking buffer for 2 hrs RT, shaking
 * 3) Wash 3 times (5 minute each) using Wash buffer
 * 4) Incubate blot with primary antibody (1:1000, ie.,10ul in 10ml of Buffer), 1 hr at RT
 * 5) Wash 3 times (5 minute each) using Wash buffer
 * 6) Incubate blot with anti-rabbit IgG (1:10000, ie., 1ul in 10ml of Buffer), 1 hr at RT
 * 7) Wash 3 times (5 minute each) using Wash buffer
 * 8) ECL solution (5+5), develop 1 min
 * 9) Drain, xpose (1min gives good bands)