OBJECTIVE: We are going to use the nitrocellulose from WB 20200304 and perform stripping to re-incubate with different antibodies. We think that there is unspecific staining using the same primary antibody for crosslinking and for detecting the protein. For there reason, we are going to change the primary antibody.
PROCEDURE:
- Stripping buffer diluted 1/5 in destilated water for 10 minutes (NewBlot Nitro Stripping buffer (Odyssey, nº 928-40030) )
- Wash with PBS-tween0’1% (3×5′)
- Blocking: blocking buffer Casein – PBS 1:1 1h
- Primary antibody rabbit (diluted 1:200 in Casein-PBS 1:1) –> 1h at RT or overnight at 4ºC
- Wash with PBS-tween0’1% (3×5′)
- Secondary antibody GAR800 1/7500 (diluted in Casein-PBS tween0’1%): 1h at RT
- Wash with PBS-tween0’1% 1x (2×5′)
- Wash with PBS 1x (1×5′)
- Read with Odyssey equipment
RESULTS: We see a band that can be sCNTN1, but we have some other unspecific bands too, as with the other experiment performed on 20200304. We are going to repeat the Co-IP with other sera, performing 2 gels, and send one the possible sCNTN1 band to mass spectometry.
