I added lysis buffer and have the protein. Should do BCA (Bradford assay). Watched so many videos but don't understand the concept or steps. I need to test say four proteins (caspase 3,9 , PC 51, cyclin d). How to prepare the sample or what info shall I know about protein? What percent of BCA buffer? Thanks a lot.
Hi Heba,
To make sure you use/load the same amount of protein for each sample, you need to know the total protein concentration. BCA and Bradford are two different assays to measure total protein in your sample.
Based on what kit you have (I am assuming BCA) you should find information on how to do a standard curve (by making a series of dilutions of a known conc. standard protein), and what volume of BCA reagent you should use (this will depend on whether you are using plates/ or cuvettes. You will need to mix only a small amount of your sample which will also depend on how diluted is your sample. But after a first attempt you should be able to work out if your samples are within the standard curve range.
you can measure the absorbance of your samples along with your standard curve points at (I believe ~ 600 nm) in any spectrophotometer or plate reader. Then, work out your unknown samples from the standard curve.
Good luck!
I agree with the comments on how to use BCA or Bradford assay for protein quantification. However, I am still raising some points for you to think about them.
Are your proteins recombinant or non-recombinant? If they are recombinant, which expressin system have you used for production of your proteins? If they are not recombinant, are they cell lysates/tissue homogenates? What is the objective of running western blot? Are you going to compare expression level between proteins? or Are you using western blot for simple qualitative detection?
If you want to quantify your target proteins, you need to purify the target proteins from cell lysates/tissue homogenates. Otherwise, I can not see the purpose of performing BCA or Bradford assay using the cell lysate. At the end, you will have total protein concentration from the cell lysate.
If you are deciding to quantify total proteins from the lysates, you need to make sure that the components of your lysis buffer, such as additives, should not interfere with the protein quantification assay.
Good Luck!
I have same problem. after using BCA assay how much of our samples we should run for sds page electrophoresis and western blot?
I have same problem. after using BCA assay how much of our samples we should run for sds page electrophoresis and western blot?
Sahar Ebrahimi Samani It depends on your target protein. If your target is abundant then 20 microgram is optimum loading conc.
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