Band intensity is affected primarily by the amount of loaded protein and the sensitivity of the stain. Perhaps you meant the sharpness of the banding pattern?

I mean sometimes the dye front spreads out more when it enters the resolving gel and sometimes its just a narrow line. When stained, the previous one has darker bands. Does it affect how dark/light my bands are?

Also, will increasing the volume of the sample loaded increase the protein content?

Dear Fakhria

if you increase the volume but maintain constant the total loaded amount (e.g you

load 2ul of a 1mg/ml sample or 10ul of a 0,2 mg/ml sample you will not observe main differences because the signal il proportional to the total sample amout loaded and the differences in terms of volume in the well will not affect the running once the sample is compacted when pass from stacking to running gel.

Of course if you load 2ul or 10ul of the same sample and than you load 5 times more protein in terms of total amount you will observe increasing in band intensity. You have also to take in account that as all the detection methods, there is a range of protein amount whee you can observe direct proportionality between sample loaded and band intensity (Linear range) but if you overload the gel and you exit from this range you will not able to observe increase in band intensity.

So in case you would like to use SDS-page as a way to quantify you protein sample (personally i prefer colorimetric methods as BCA, Bradford or VU absorbance at 280), load several amount of the sample and be sure that you are in the linear range. You can check which is this range just loading different amount of a protein standard as BSA or Lysozime.

good luck

Manuele

Thanks Manuele. I will increase the concentration of my sample then.