I am running Phostag gels. I finally have the system working but the concentration of my antibodies gives me a high signal. The film developed for a 1 second exposure is too intense. I am using a 1:5,000 for both the primary and secondary antibody. The Phostag gel blot seems to need a more concentrated antibody than a regular SDS PAGE gel. I am inclined to dilute the secondary to 1:10,000 and leave the primary antibody alone. Any suggestions or advice?
With many antibody-related techniques it usually seems to be a question of specificity vs. sensitivity. Of course, you want to have both. I tend to recommend further dilution of the primary antibody, but you may test several dilutions of both to get the optimal conditions for your settings.
I've not used the phostag system, but I've encountered this problem with standard SDS-PAGE gels before and have taken two approaches: load 10-fold less protein or (if you're also wanting to detect other less abundant proteins on the same blot) optimise the primary antibody concentration by testing a two-fold dilution series starting at 1:5000. This might be a bit fiddly, but you'll end up with the optimal primary antibody concentration. You could do the same with the secondary and leave the primary, but given that the latter are usually more expensive, it makes (economic) sense to optimise the primary antibody concentration. (Hope this answers your question - your third sentence seems contradictory with the rest of what you say).
Well sometimes the endogenous expression of a protein also matters. So if you are trying for any particular protein try also look for its expression level in your model system this might also help in better and quick optimization in your protein loading amount and antibody dilution. Though I have used this for normal SDS-PAGE gels, hope this might hold good for your condition also.
Hi Evelyn,
we normally use in the lab a block titration experimental design where both primary and secundary antibodies are diluted simultaneouly. This approach provide us with the precise dilutions for both reagents of the immunoassay.
Hi, Robert,
considering what Evelyn described, I think so. In our hands, the best way to optimize the WB to avoid the type of noise described is to carry out a block titration experiment, by diluting primary and secondary antibodies simultaneously.
We normally also include in our experiments controls for all secondary antibody dilutions, depending on the source of whole cell extracts that have being used in Phostag gels.
Thank you everyone for great advice. I fixed my problem by loading less protein, and keeping the antibody dilutions the same. Another question I have is reusing primary antibodies. Is it better to add Sodium Azide when they are stored in 5%TBS-T after the first use. A very naive question is how long can you keep a 10ml liquid stock of 10% Sodium Azide and what is the best storage conditions.
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