questions about secondary antibody - (Nov/26/2006 )
Hi,
I am using HRP-Goat Anti-Mouse IgG (Gamma) as the secondary antibody in my western blot experiments. (My primary antibodies are from mouse.) I found that the secondary antibody could bind to certain proteins even without the prior binding of primary antibody. For example, I loaded seven protein samples (total protein isolated from human cell lines), three of which have carboxy-term His tag. After incubation with the primary antibody (anti-His tag), only three lanes should have bands while the others should be blank. But all lanes gave exactly the same bands. By "bands", I mean there are multiple bands in each lane. I am not sure if it is the problem with my seconary antibody.
It should not be the problem of the primary antibody, because 1) this has been observed with 3 different primary antibodies; and 2) a lane loaded with control protein showed only the control protein, but not the multiple bands observed in sample lanes. (But the control protein should not show up 'cause the primary antibody does not target that protein.)
I hope I have stated clearly. I am very confused now. I just noticed that some companies suggest to protect the HRP-conjugated secondary antibodies from light. I did not do that 'cause the vendor I used did not say so in the product information sheet. Could that be the source of the problem? Can anyone suggest a anti-mouse secondary antibody that shows very clean bands? Any advice or suggestion to my problems would be really appreciated!!!
I am using HRP-Goat Anti-Mouse IgG (Gamma) as the secondary antibody in my western blot experiments. (My primary antibodies are from mouse.) I found that the secondary antibody could bind to certain proteins even without the prior binding of primary antibody. For example, I loaded seven protein samples (total protein isolated from human cell lines), three of which have carboxy-term His tag. After incubation with the primary antibody (anti-His tag), only three lanes should have bands while the others should be blank. But all lanes gave exactly the same bands. By "bands", I mean there are multiple bands in each lane. I am not sure if it is the problem with my seconary antibody.
It should not be the problem of the primary antibody, because 1) this has been observed with 3 different primary antibodies; and 2) a lane loaded with control protein showed only the control protein, but not the multiple bands observed in sample lanes. (But the control protein should not show up 'cause the primary antibody does not target that protein.)
I hope I have stated clearly. I am very confused now. I just noticed that some companies suggest to protect the HRP-conjugated secondary antibodies from light. I did not do that 'cause the vendor I used did not say so in the product information sheet. Could that be the source of the problem? Can anyone suggest a anti-mouse secondary antibody that shows very clean bands? Any advice or suggestion to my problems would be really appreciated!!!
You need to use different dilutions of your antimouse IgG. Non-specific binding disapprears when it reachs certain dilutions while specific does not.
you should always determine by yourself the dilution of the secondary antibody.
the provider of mine says 1:5000 and I use 1 :20 000.
make a blot with your cell extract in all the lanes (all lanes are identicals), block the membrane and cut strips, incubate the strips with increasing dilution of the secondary antibody.
reveal.
the best dilution will be the lowest dilution that doesn't give a signal (non specific signal).