Hello Mateen Noor

The Bradford assay is used to determine protein concentration of the sample for gel electrophoresis.

For Bradford assay, a set of standards is created from a stock of protein whose concentration is known. The Bradford values obtained for the standard are then used to construct a standard curve to which the unknown values obtained can be compared to determine their concentration. Use a protein as your standard that most closely resembles the protein you are assaying. BSA and IgG are typical standards used to construct the curve.

Prepare a standard curve of absorbance versus microgram protein and determine the concentration of the unknown sample from the curve.

If you have diluted your sample in order to fit in the linear range of the curve, then you should multiply the protein concentration of your sample obtained from the curve with the dilution factor to get the original protein concentration in the undiluted sample.

When you load your protein sample on to the gel, it is recommended that you load in terms of concentration and not in terms of volume of protein. Generally 10-20ug of protein per well is loaded on SDS PAGE. You may also load to a maximum of 30-40ug protein per well.

However, loading volume is dependent upon the type of comb used (i.e. well thickness and length) and thickness of the gel.

You may want to refer to the link below for more details on the volume of protein to be loaded on the gel.

https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/recommended-well-loading-volumes-sample-loads.html

Best.

You typically want to load equivalent total protein (Ptotal) amounts in the wells of a gel. You run a protein assay to get the concentration of protein in the sample (Cprotein). A simple material balance can be solved to get the loading volume (Vload) required.

Ptotal = Cprotein x Vload => Vload = Ptotal/Cprotein

Note, you have to track of the units for each term.

Hi Mateen Noor, with Bradford assay you get the concentration of samples, which is represented as mass/volume (c = m / v). So, you must establish the mass of protein to interrogate in your experiments. For instance:

Conc. of sample = 2ug/uL

Mass to interrogate = 20ug

If v = m / c, thus

Volumen = 10uL