What reagent do you use to dilute your cell lysates after finding protein concentration with Bradford assay? Do you use 1X loading buffer or do you use water?
If you use the 1X loading buffer to dilute, do you then add more loading buffer to your sample before you boil it? Ex. 50 µL 4X loading buffer in the original 150µL cell lysate and then you dilute to the proper amount with 1x loading buffer.
150µL cell lysate will take 150/ 5 ( For 5X loading buffer ) i.e. 30 ul loading dye or 150/4 ( For 4X loading buffer ). 150 is practically impossible to load in 1 or 1.5 mM thick gel. You can dilute your protein in the buffer in which you made cell lysate. Since 50 μl is the maximum loading volume, the sample must be > 4 mg/ml protein. Loading 50 μg is also fine for coomassie stain and 15-20 ug for western blot
Mahesh Chandak so if I used RIPA buffer to lyse my cells, I would use that to dilute?
RIPA buffer has substantial absorbance in the UV, and your estimated protein concentration is wrong. Yes, you can use RIPA to dilute your samples not to measure protein concentration.
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