I have quantified the lysate by bradford method and loaded 25ug of lysate for SDS PAGE and proceeded for western blot. However, upon detection I see a variation in loading control i.e., GAPDH. I am sure there was no issues while loading the samples in the gel. How can I resolve the issue to get even loading controls for my samples?
If you used BCA/Bradford method to quantify protein, you've shown that you loaded equal amounts of total protein. However, individual protein expression can be varied. Loading control proteins such as GAPDH, Beta-Actin, Tubulins (and others) are only suggestions as a beginning point to test out, never assume any protein is stably expressed in your conditions. You need to check to see whether your loading control is actually stably expressed. I know from my PhD studies, GAPDH was stably expressed in one condition but not in another condition.
@Can keissleng, thanks for the suggestion. I am using Lipopolysaccharide treated samples for one of my conditions. Can you give more insight about suitable loading controls for LPS treated samples?
You could check with a quick and reversible (just wash with buffer) Poinceau S staining of the blot membrane before blocking to see the lanes and judge if you loaded similar amounts of protein. if you see no significant differences then your housekeeping genes may not be that stable in their expression under the experiment conditions.
Hello, that is exactly what you need to determine in your experimental condition(s). If someone has previously published/observed that GAPDH is stable in the same conditions that you are planning your experiment on, then it's a good start to use GAPDH and see how it works out for you after you get the result. In your case, if you are comparing LPS-treated cells vs placebo/non-treated cells then do a reference protein study with 2-3 loading controls and see if their expression is similar between those conditions (compare intensity). Pick reference proteins that you feel will be appropriate for your experimental parameters, sometimes people pick loading controls specific to their target's protein location i.e. cytosolic normalizer for a cytosolic target protein (see table 1 - https://blog.cellsignal.com/choosing-a-western-blot-loading-control-cst-blog). Depending on their weight, you may be able run 2-3 loading controls if they are separate enough. Then compare intensity and draw conclusion(s). Honestly, if you could I would recommend you look into total protein staining to quantify as it is gaining increasing traction and less of a hassle than this business of finding stable loading controls - https://www.licor.com/bio/reagents/revert-total-protein-stain-for-western-blot-normalization
Thank you @Can and @Huseyin for the insights. I have tried doing commasie staining of the gel as well as ponceau but the bands look almost similiar. Issue arises while visualizing after probing for western. However, I will try finding appropriate loading control and total protein.
I just checked my old PhD data from 2013 in mouse hearts, going by intensity GAPDH was more abundant in 48-week old mouse vs 8-week old (female) mice. You're not the first to experience this, and you won't be the last :). That's why total protein normalization is the way to go in the future!
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