User:Vijay/Protocol/western-msa

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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)
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