I want to detect phosphorylation signals (pAkt, p-p38, pERK, pJNK, p65, etc) in T cells after being stimulated with hamster anti-CD3 and hamster anti-CD28, then began to calculate time after adding goat anti-hamster IgG. There is a annoying IgG band (about 50kDa) on wb membrane.
Who can tell me how to get rid of the IgG band?
You may try to preclear your lisate prior to WB by using Protein G Sepharose beads. I have used them before in tissue lisates to eliminate Ab contamination from blood and it worked just fine.
You could use antibodies directly labeled by HRP (e.g. abcam is selling Anti-AKT1 (phospho S473) antibody coupled to HRP)
Use a secondary antibody that does not detect denatured form of IgG. Most companies sell these now.
Don't add betaME in to your sample buffer, it will not break the heavy and light chains.. you will expect to see a single band at 110kD this way you can avoid the 50kDa light chain in your blot.. good luck
It is the secondary antibody picking up the heavy and light chains in your lysates. So, your secondary will have to be non-reactive to the endogenous immunoglobulins in your lysates. If you are looking at human lysates, you will be fine using secondaries with minimal cross-reactivity to human serum proteins, but the best solution is to avoid secondary antibody all together and go for direct conjugated primary. We find biotin a good solution in such cases and go in with streptavidin-HRP as the secondary.
It is likely that your secondary antibody was cross reacting with the hamster IgG or goat IgG. Getting a secondary antibody which recognizes light chain only will solve your problem.
Depending on the amount of IgG present in your sample, you might face problems with proteins saturating the membrane at the respective sizes, so there is some risk that you underestimate the amount of phosphorylation or the readout is influenced by the actual amount of totatl protein loaded. You should at least rule that potential problem out by spiking some samples with appropriate IgGs and measuring their influence on the phosphorylation signal.
Thus, I'd prefer removing competing proteins before running the gel, e.g. by Protein G beads or simply snippets of NZ oder PVDF membranes saturated with protein G (and blocked afterwards to be safe) or appropriate spin columns or by some other way in the process.
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