Hello, All,
I have been trying to optimize my western blot which has been giving severe background issues since many days. I have tried to increase the wash time, even then the background persists. I am using a PVDF(0.2 micron) membrane. I have been asked to try using nitrocellulose to see if that background is eliminated, but I would want to also perform a stripping, which I feel is very harsh for a nitrocellulose membrane. Is there a better alternative or any suggestion on optimizing this?
Thanks,
Do you think the type of membrane will be the main cause for your problem? Many factors can contribute to your western blotting problem. Thus, it is good to consider the effect of all potential parameters, such as gel, protein sample, buffers, membrane, transfer method, blocking solution and blocking time, washing solution and washing frequency, purity and concentration of Abs, incubation time and washing frequency, others....
Hello Sneha. As mentioned by Dr Tekewe above, there are many factors that can contribute to a less-successful western blot. Is it possible to share an image of your blot? Maybe we can start troubleshooting from there.
Hello, thank you very much for your responses. I have been using 5% non-fat dry milk as my blocker and perform blocking for 1 hour at RT. The antibody that I use is a monoclonal antibody.
Dr.Nurul Adhwa, yes that sounds like a good suggestion. I will upload the images soon, so that you can better suggest on what exactly the problem is and how I can go about from there. Thank you very much!
PVDF is going to endure stripping and reprobing far better than nitrocellulose. As far as blocking, 5% dry milk performs the best among the blocking agents that I am familiar with when it comes to reducing spurious background, but it also reduces signal intensity of your antibodies of interest. Let's see your blot images first.
I don't think your problem comes from the type of membrane.
I usually strip nitrocellulose membranes with no major issues...
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