How can I get uniform bands by western blot? I try so many times with the same sample but I didn't get uniform bands (even for beta actin too).. I check the calculations for protein concentration but in my mind it is okay.. if any body has experience or they calculated by any reliable method please share with me. I used BCA/coomasi blue to find out concentration.if calculation is in excel sheet will be appreciated. i am going to attached the file of mine calculation.
Hi Farhan,
I see some mayor problems in your protein assay approach:
1. the absorption of your unkown samples are equal or higher than your highest bsa-standard concentration.
2. the curve for bsa standard looks like the assay is reaching saturation at or even before reaching 2µg bsa (but you can't tell --> see below)
3. you should use more samples for bsa standard. You only have 3 samples really containing bsa (0,5µg/µl, 1µg/µl and 2µg/µl), so I would recommend to use 0,125µg/µl, 0,25µg/µl, 0,5µg/µl, 0,75µg/µl, 1µg/µl, 1,5µg/µl and 2µg/µl. Then a possible saturation of the assay system is easier to detect.
So, never use absorption values higher or ,lower than covered by your bsa standard curve!!!
A easy method is simply to dilute your samples 1:2 and 1:5 (optional 1:10) and do the assay again. If all obtained absortion values are within the linear range of your assay a recalculation of the diluted sample should yield comparable results!
Best,
Kai
Thank you so much Doctor for your kind suggestion, i will try with your suggestion.
Basically the amount of BSA you use for protein estimation does not affect the uniformity of band, it seems there is fundamental mistakes in your calculation. I think you have problem in gel making and loading of sample for electrophoresis as well.
additionally to some previous comments:
- std curvre (0 and other protein concentrations) must be done in same buffer concentration as samples. (means you need to add your lysis buffer in the std curve at same final concentration that it is in you samples-BCA mix
- Also, depending on your cells and exp conditions you could affect how your total concentration reflect the reality comparing to actin.
Considering you want to know samples real values for a given protein, and have difficulties, Try to check samples without any manipulation. the variability of the paralells will tell how your actual method works. All of the above suggestions could help, of course. But try to measure 5 or 10 or 15 parallel samples on the same blot.
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