I have done a standard curve for protein using BSA (absorbance vs ug/ml). How do I calculate the amount of protein in the cells in mg/g FW? I extracted 0.5g of cells in 2ml buffer and used 0.01ml for protein estimation. tq.
The way you measure the protein concentration of your cell extract is the same way you have done your standard curve. Following is the method I use. I use 0,5 10,15 and 20 micrograms of BSA in the various samples for my standard curve. I do this my making a 1 mg/ml solution of BSA and then Take 100 micro-litres of water (for blank) and 95 water + 5 micro-litre (BSA), 90 water+ 10 ul (BSA), 85 water+15 ul BSA and 80 water + 20 ul (BSA) for the rest of my standard curve samples. I add 1ml of 1X Bradford Reagent to these for color development. For my experimental sample (the cell extract) I prepare two aliquotes of the extract, one of which is 5 times diluted and the other 10 times diluted. So we have 3 experimental samples which we name 1X (original extract), 5X (5 times dilution) and 10X (10 times dilution). So use 2 ul of each of these solutions and and 98 ul of water and 1ml of bradford to get 3 experimental samples 1X,5X and 10X. Take the OD (at 595nm) of each one of the samples. Use the OD of the samples with the known protein concentrations to plot the standard curve (assuming the OD of 0 BSA as the blank) and use an MS Office Excel program to plot the graph and use trendline option in that program to check out the equation of the line. The equation shall be y-(some number) * X where y is the OD and X is the amont of proteins in micrograms. Use this equation to find the amount of protein in your experimental samples, and thats what is in the 2 ul you have added. So you have 3 values of the protein concentrations for your cell extract. Take an average of the three readings!! You should however ignore any experimental reading if its much higher than the 20 ugm reading of the standard curve and take into account only those readings which fall between 0 and 20 ugm reading. This is done to keep the reading within the linear range of bradford. Hope this helps!!
Hi Jaideep Mallick, thanks for answering. I've a standard curve and done with my sample. For example, my sample is 2 ug/ml, how to I convert the unit of 'ug protein / ml' into 'mg protein /g Fresh Weight of Cells'? I extracted 0.5g of cells in 2ml buffer and used 0.01ml for protein estimation. Thank you.
Thats not difficult. See once you know the protein concentration of your sample, measure the total amount of lysate. Say you have 2 microgms of protein per microlitre (as from the calculation I have shown above you shall have the result in micrograms in microlitres) . Then you multiply with the amount of lysate (say for example 2000 microlitres) to get the total amount of protein from your sample. Now say you had 0.5 grams of cells to start with ( which is the weight of your pellet before you started breaking cells) then you have 4000 micrograms of proteins / 0.5 grams of cells. So that means you have 8000 micrograms per gram of cells which means 8 mg/gram of cells. Hope this helps.
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