IP and western blot - (Aug/11/2009 )

I normally do IP then western blot but my problem is i got an extra band (my band in 52KDa and the extra band 30 KDa)
i followed thr folowing protocol
1- add 5ul of mouse anti human antibody (IP) incubation over night
2- add 20 ul proten G agarose
3-collect immunoprecipitates then wash pellet 4 times with PBS
4- dilute pellet with 30 ul lysis buffer and add sample buffer , boil , centerfuge to pellet the agarose beads
5- SDS page analysis of the supernatant
6-Transfer to nitrocellulose membrane.
7- blocking
8- incubate membrane with mouse anti human antibody( primary)
9- wash the incubate with rabbit ant mouse antobody HPR (secondary)
10- wash
11- ECL
12- X ray film
please help me by answer me why i got an extra band?????

many thanks

Usually this happens when you expose your membrane for too long. Try exposing it for less time. Are your bands very dark? Also this could be due to non-specific binding if your antibody dilutions are too concentrated.

the exposure time is very short
and regarding the concintration of the AB isit for IP or WB???

inas on Aug 12 2009, 05:03 AM said:

A picture of the blot would help. Do you see this band only in the IP lanes and not in the input lanes on your blot? If so, it is likely to be the antibody light chain, which comes off the protein G beads with your target proteins when you boil your IP samples.

thank you very much for your reply
actully i couldnt understand the mening of your statment, could you please explane more for me.
regards

Your 30 kDal band is probably the light chain from your IP antibody. Do you ever see a doublet around your 50 kDal band? I wouldn't be surprised (with your antibody combo) if you were also picking up the heavy chain from your IP. Typically, if you are doing an IP and you see a 25-30 kDal band and a 50 kDal band, it is from your light and heavy chains of the antibody.

Do you see these bands in your isotype control IP? If so, they are almost definitely the light and heavy chains.