Problem with protein quantification - (Jan/13/2009 )
Hi, I am new to proteomics
I am now trying to use BSA as standard to plot a calibration curve. I use the Bradford method (from Bio-rad quantification kit) but I have some problems;
1. I reconsitute the lyophilized BSA using double distilled water (is it better to use lysis buffer to recobstitute it)?
2. I don't know what should be the ingredients for the reference in spectrophotometer cuvette - is it water + lysis buffer + dye + HCI (optimized for quantification in lysis buffer)?
3. Normally how many standard data points u use to make the calibration curve, and what r the concentrations of the BSA standard u use in plotting the curve?
Hope not too long. Plx give me some advice. Thx in advance.
I am now trying to use BSA as standard to plot a calibration curve. I use the Bradford method (from Bio-rad quantification kit) but I have some problems;
1. I reconsitute the lyophilized BSA using double distilled water (is it better to use lysis buffer to recobstitute it)?
2. I don't know what should be the ingredients for the reference in spectrophotometer cuvette - is it water + lysis buffer + dye + HCI (optimized for quantification in lysis buffer)?
3. Normally how many standard data points u use to make the calibration curve, and what r the concentrations of the BSA standard u use in plotting the curve?
Hope not too long. Plx give me some advice. Thx in advance.
for question 2:
are you talking about the control background of the spec. ?
if so, it depends on what you will test later
for question 3:
I usually takes 6 points
If you are reconstituting BSA in water, I think its fine, why would you resuspend it in lysis buffer? and for your second question I agree to Dannychow, it'll depend on what will you test later.For example when we do it for lowry assay, we take blank which has everything except protein and many times for some other methods we take water as blank. we also take 5 to 6 points to plot a standard curve.