I was mixing my secondary antibody in milk + TBST when I realized that I blocked with milk + dH2O. What happens when you block for a Western with milk + dH2O instead of milk + TBST? Mostly wondering if it's even worth finishing the secondary antibody incubation and film development or if I need to start over by stripping it and redoing the blocking and antibodies.
I don't think it should have a huge bearing on the experiment other than giving you a smeary blot. You've nearly finished it already, so unless you need a superbly crisp blot for a publication, I would recommend you go ahead and finish it. If the blot looks ok and shows you what you want to see after developing it: HURRAY!!! You're done. If it looks bad: you will still be able to strip it and start all over again.
I don't think it should have a huge bearing on the experiment other than giving you a smeary blot. You've nearly finished it already, so unless you need a superbly crisp blot for a publication, I would recommend you go ahead and finish it. If the blot looks ok and shows you what you want to see after developing it: HURRAY!!! You're done. If it looks bad: you will still be able to strip it and start all over again.
Hi ,
From our experience, it will not affect your results. Some people in our lab use plain tap water to wash their western blots, and it still works. You might get a little bit more background because there was no detergent present during antibody incubation, so maybe add the detergent in the washing buffer.
I agree with the others, if the blot looks ok go with it. Might have a little higher background due to lack of tween.
Like the others have stated, it shouldn't have too much of an effect other than giving a little more background. Since you're so close to the end of the Western already, just go ahead and do the developing and see how it looks.
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