I would like to know what is the difference and, the advantages and disadvantages of loading equal volume of cell extract instead of loading an equal amount of protein per lane in SDS-PAGE. I am planing to compare band density using Western blot band densitometry following normalization for beta actin loading in both cases.
Ideally cell number under the study were comparable in all cell culture dishes at start by seeding equal cells in dishes which received different treatment. Those treatments are expected to affect cell growth, viability and/or alter given protein synthesis.
Actually there is not a particular advantage. If your treatments are as you say expected to affect protein synthesis, you will still need to have a total protein reference. Starting with a roughly equal amount of cells will make sure that the protein concentrations of all of your samples will be within a very narrow range, but still for quantitative work it would be preferable to load equal amount of proteins than extract volume. This is particularly important because housekeeping proteins like tubulin, actin, GADPH etc are invariably indifferent to experimental conditions and will remain roughly stable in an equal protein load. Hope this helped
Depending on what kind of sample you have its not easy to take a certain amount of cells. Especially when you start with tissues its easier to use the total protein amount or the amount of a calbrator protein (like beta-actin, beta-tubulin).
The whole point of loading equal amount of protein is to see if the differences you are having, if any to occur, are because of difference in expression in response to treatment, or was it because you have loaded different amounts of protein. Thus, using equal amounts and proofing that by measuring actin, or GAPDH or any other loading control gives credibility to work.
Loading of equal amount of protein is of utmost necessity than loading equal volume of cell lysate, because different samples of equal volume will have different protein concentration, therefore the effect of treatment will not be clearly understood, even normalization is done by the loading control, as loading controls are generally stably expressed than the other proteins. Even plating equal amount of cells doesnt guarantee equal amount of protein in equal volume of samples, because different proportions of adherence will be there and different amount of cell death will happen. So its always better to go for protein estimation as it reduces the effort of band comparison after normalization. Hope this helps !!
Best of Luck !!!!!
Thank you all for your kind response and assistance. I would like to know please how to distinguish whether the expected change in protein expression is due to actual inhibition and not due to cell death or slow growth.
Thank you Subhadeep Chakrabarti for your kind care and assistance. I would like to know please whether normalization for difference in the B Actin can be achieved by loading the same protein amount in each lane i.e., different volumes of CE or is it better to load the same volume then normalize for loading control densitometery by dividing each band density by the ratio of its beta actin to the beta actin of the control?
Thank you
when normalizing data, should I divide each band density by the density of its beta actin in the same lane or I could divide beta actin in each lane by the beta actin of the control lane then divide the density of each band of interest by the correspondent ratio of its beta actin to the control.
Thank you
If the density of the actin band in the control is greater than the density of the experimental situation, your adjusted density of band of interest would go higher. You would want to use the following equation:
adjusted density of band of interest in experimental = actual density of band of interest in experimental X density of actin band in control / density of actin band in experimental. You can change the equation accordingly if the density of the actin band in the control is lower than the density of the actin band in the experimental. If the densities of the actin bands are different by 10% or greater, however, I would re-do the experiment.
I have a question in that regard,
I pipetted two different volumes of different protein concentrations in SDS-PAGE
Sample 1=
The obtained protein concentration is 1.25 μg/μl and 5 μg was needed
5/1.25 = 4 μl
Laemlii puffer =3.75 μl
So, 4 μl + 3.75 μl= 7.75 à 15-7.75=7.25 from the lysis buffer.
15μl, but the actual amount that has been loaded is 5μl only
Sample 2=
The obtained protein concentration is 1.25 μg/μl and 2.5 μg was needed
2.5/1.25 = 2 μl
Laemlii puffer =3.75 μl
So, 4 μl + 3.75 μl= 7.75 à 15-7.75=9.25 from the lysis buffer.
15μl- I loaded here 10μl to the gel well
My question is that they migrated the same distance! I can't find a scientific explanation for that, please if someone has any clue, clarify my confusion.
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