To determine protein concentration of unknown samples (whole cells lysate) coming through the standard curve that has to be plotted before, I know that that can be obtained after multiplying the diluted samples concentration by the dilution factor (used to dilute samples), after that dividing the desired concretion by the sample concentration.
On the contrary, how can I calculate the concentration of undiluted samples? Meaning if I don't dilute my samples and add a certain volume of them as they are directly into wells as there will be no dilution factor..
I am asking this question because when I made protein determination by BCA method, I got low proteins quantitation in my samples when they have been diluted by 1/4. However, I did the opposite latter as I added 10 ul of each original sample (undiluted) per well and later mixed 10 ul with 200 ul of BCA reagent , then I got appropriate protein concentration but I don't know how to calculate the next step; as I am going to load 25 ug of protein per well of SDS-PAGE. My loading buffer is 2x SDS-Page.
Therefore could anyone please suggest something or give me an example to know how to do that?.
Many thanks,
Prepare a calibration curve with BSA by plotting A280 absorbance values vs microgram conc of protein BSA with final volume in each case 1 mL. Then determine the A280 value of your protein (10 microlitre) dissolved in 1 mL buffer. Use dilution factor 10 and represent final protein conc from the reference curve of BSA. UV Visible spectroscopy is a relative method as you need a calibration curve!
Thank you very much Shamsher, but could you please clarify that more as I am still confusing with the written calculation. Knowing that I use BCA method (standards range: 0.2-1.6 mg/ml) to determine protein cons and I did not dilute my samples, just I added 10 ul of original ones directly into 96-well plate and thereafter the 10 ul volume inside each well was mixed with 200 ul of BCA reagent.
Many thanks,
So long as the final volume in each case is same, the concentration can be directly obtained from the reference curve.
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