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)