3 replies to this topic

[joeychuk]

I'm doing Co-IP lately to detect the interaction between two proteins, protein A is about 50kDa and protein B is about 100kDa. I first used anti-protein A in IP and then anti-B for WB. On WB, it supposed to have a 100kDa band but I detected a strong 150kDa band instead.. What's wrong with my results? I used SDS-PAGE gel and boiled the protein before loading. I use non-reducing buffer as the loading buffer. Thank you for your help!!

Joey

[amelia417]

joeychuk, on Feb 26 2010, 11:53 AM, said:

I'm doing Co-IP lately to detect the interaction between two proteins, protein A is about 50kDa and protein B is about 100kDa. I first used anti-protein A in IP and then anti-B for WB. On WB, it supposed to have a 100kDa band but I detected a strong 150kDa band instead.. What's wrong with my results? I used SDS-PAGE gel and boiled the protein before loading. I use non-reducing buffer as the loading buffer. Thank you for your help!!

Joey

If you're not fully reducing the interaction of A and B, you might be detecting A and B as a combined MW of 150 kDa. To prove whether or not this is true, you should in theory (assuming that the antibodies against each A and B, respectively, still can still interact with their epitope(s) with A and B are interacting) detect the 150 kDa band with the antibody against A AND with the antibody against B.

Why not try a reducing buffer in one lane and a non-reducing buffer in another lane for two otherwise identical co-IPs and see if the 150 kDa band disappears under reducing conditions and results in the 100 kDa band for anti-B?

[joeychuk]

Thank you very much, amelia417! I'm actually trying the regular WB with anti-B with reducing and non-reducing buffer right now.

Another question is, is there any protein modification that would change the size of the protein on a WB blot?

[laurequillo]

joeychuk, on Feb 26 2010, 06:43 PM, said:

Thank you very much, amelia417! I'm actually trying the regular WB with anti-B with reducing and non-reducing buffer right now.

Another question is, is there any protein modification that would change the size of the protein on a WB blot?

There are some (Sumoylation and phosphorylation for example) but not from 100 to 150kda!!  

are your proteins endogenous proteins or everexpressed ones?