I am working with cell lines. Usually, I have to extract total protein as cell lysate and run SDS gel. I want to know what protein concentration is ideal for western blot analysis to load into SDS gel as cell lysate.
Ideally, for cell extracts, 10-50ng of protein lysate should be loaded but that will depend on the expression of your protein of interest. Also, it would be better if you do a titration, that would give you an idea of how much to load on the gel.
I would suggest checking your protein concentration in cell lysate by Bradford assay and then doing accordingly. Usually, a 60ug protein concentration is enough for western blot per well. You have to calculate the volume per your protein concentration from the different groups. Please keep the protein concentration for loading constant for all wells. Linchi Rani
Hello Kamindu Gayashan
Generally, for a cell lysate containing the protein of interest, 10-20ug of protein per well should be enough. If you are interested in low abundance protein in the lysate you could increase the protein concentration to 30ug per well. However, for purified protein you may load 1ug of protein per well. Do not load excess protein as it may lead to the formation of non-specific bands in the final results.
Usually, proteins are loaded on the gel in terms of concentration and not volume. However, loading volume is dependent upon the type of comb used (i.e. well thickness and length) and thickness of the gel.
You may want to refer to the link below for more details on the volume to be loaded on the gel.
https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/recommended-well-loading-volumes-sample-loads.html
Best.
The ideal protein concentration to load in an SDS-PAGE gel for western blot analysis can vary depending on the specific protein of interest and the sensitivity of the detection method used. Typically, loading between 10-30 µg of protein per lane is a good starting point. However, it is important to optimize the amount of protein loaded to ensure that the signal is strong enough for detection but not so high that it becomes saturated or overexposed. To determine the optimal protein concentration, it is recommended to run a series of gradient gels with different amounts of protein loaded per lane and perform a pilot western blot to assess the signal strength.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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