We extract protein from tissue or cells, quantify the lysate and then load equal amount of protein on the gel for western blotting. However, it is common at times to observe unequal band intensities for housekeeping/normalising proteins like GAPDH, actin etc. Since we use these normalizing proteins as basis for interpreting the change in levels of target protein upon probing with the antibody, How important is the protein quantification part? Can't we avoid protein quantification by using a fixed amount of laemmli buffer based on cell number or density, immediately denature and load equal volume on gel, stain the gel and assess if lane intensities are equal or not, and thereafter use the sample for western. It requires a bit of practice but we observe equal intensity for normalizing proteins. Would like to have western blotters' feedback.
Yes definitely. Protein quantification is must for the western blot analysis because of it give you exact fold expression of your targets. Without quantification of proteins also give the precise results when normalized with housekeeping protein of same, it will give you good results either up or down regulation of your targets only, but the fold of protein expression is slightly differed between the same amount of protein loaded gel and without quantified protein loaded wells after normalization. My suggestion is to quantify the proteins and then do the western blot analysis.
It depends up on the objective of running western blot. If you are running western blot for comparison of expression levels between different constructs, you should quantify the proteins prior to running western blot. However, if you are running western blot for qualitative detection of protein expression from cell lysates/or tissue homogenates, it may not need to quantify the proteins, rather you can load the same volume.
So, one key thing to remember is: Western blot is not quantitative. It is, at very best, semi-quantitative. But you can get very different quantitation simply based on how much protein you load, how strong your antibody is, or how long you develop your blot.
Regarding housekeeper genes: one normalizes to those because it's better than nothing. But some cell lines will express different relative amounts of, say, actin than others will. So they're not 100% reliable as a normalization mechanism. Overall, you'll probably have the most luck with peer review if you have at least one housekeeper gene being blotted, but most people won't care which one you use, so try a few, and if any of them look good in your system, use that one.
As for your very first question: loading based on total protein amount is the most common way to do it, but it's not necessarily better than any other, non-random loading protocol - it's just comparatively simple and relatively reproducible. You could just as easily load total protein from X cells, or do as you seem to be doing here, and load an amount of protein that gives you equal expression of some standard protein like tubulin. The key here is to be able to provide a simple explanation of what you did and why you did it in such a way that others can reproduce your work, and in such a way that your interpretation of results is reasonable.
If you load X ug of protein, you are saying that for every gram of total protein, your cell has Y grams of your protein of interest. If you load X cells of protein, it's every cell has Y grams. If you normalize to actin, it's Y grams of protein of interest per gram of actin.
The first is, again, most common, probably because it's simplest. But is it better? Depends on the question you want to answer.
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