Dear Ahmad El-Hamamy ,

first of all there is no such thing as a stupid question, only stupid answers. Better to ask and taking a risk of appearing stupid than to continue making stupid mistakes or working thoughtlessly.

So I have Crystallized BSA as a powder form, how do I work with that? How to make a solution, and how much NP lysis buffer, water and BSA to prepare for that?

You have to resuspend your BSA to a starting concetration (for example 1 mg/ml or 2 mg/ml) and further on you will dilute your stock to prepare a standard curve. You should choose a buffer by yourself because there is so many factors which have to be taken into account. However, BCA kit always contains a multiple stocks of BSA already resuspended in concentration of 2 mg/ml. So, I guess you should not worry about it.

Another question, during the BCA assay, in the 96 wells, how much water and lysate per well? Some protocols say 2 ul lysate and 18 ul water, other protocols with other colleagues say 23 ul water and 2 ul lysate. I tried both on different days, is there a difference, or it doesn't matter?

It does not matter. You just need to remember that appropiate volume should be added to your standard curve. Personally, when I use 96 well-plates I add 2 ul of lysate and 8 ul of water, in case of eppy tubes still 2 ul of lysate and 98 ul of water.

Best,

Michal

• Dissolving BSA is not trivial, as it forms a gluey mess upon contact with water. An end-over-end mixer overnight works, however.
• Weigh out, say, 10 mg of BSA (and a preservative like sodium azide or thimerosal) into a 15 mL screw-top tube, add water to the 5 mL mark and rotate it overnight in the cold room.
• Transfer the solution to a 10 mL volumetric flask, rinse the tube by vortexing 2 times with 1 mL water and add that into the flask too.
• Carefully add water to the 10 mL mark to give a 1 mg/mL weight/volume (w/v) stock. A Pasteur pipette is good for this.
• Freeze aliquots of the stock at -20 °C, each aliquot should be enough to last you for a week or so. The aliquot you are working with should be stored on ice or in the fridge, do not re-freeze.

You can, of course, buy ready-made stock solutions as well, control serum is a 70 mg/mL solution of BSA in buffered saline that is used in clinical chemistry as blank and relatively cheap.

• During an actual assay, use several wells per sample, adding 0, 2, 4, ..., 20 µL of protein solution and 20, 18, ..., 0 µL of water.
• The readings plotted against the volume should be a straight line (use linear regression after plotting the original data to check for linearity).
• The slope is proportional to the concentration. In other words, you can use the rule of three to calculate the concentration of your sample from the concentration of your stock and the slopes of both sample and stock.
• By the laws of error propagation, the sum of the relative errors of the slopes is a worst-case estimate of the relative error of your protein concentration. With common lab equipment and some practice, the error of the slopes should be less than 2%.

Ahmad El-Hamamy, I just want to list some basics in addition to what colleagues said here.

When working with BSA powder;

-Resuspend your BSA in whatever the buffer your samples will be in. By doing this, you will eliminate any color change coming from the buffer.

-Prepare high concentration BSA solution like 20 mg/mL. Then dilute it two fold every time and go from 20 to 10, 5, 2.5, 1.25, 0.625 etc. Use 20 mg/mL to prepare 10, mix very well and then use 10 mg/mL to prepare 5 mg/mL. It goes on like this.

When doing BCA assay;

-18 ul or 23 ul water shouldn't make a difference. Just be consistent with what you do all the time.

-Another point to remember, do not forget to do a blank sample. BCA reagent with buffer your samples and standards are in. You will subtract this from all of your measurements.

Just a grammar note for all you folks. The term "resuspend" mean that you return the sample to the original volume; therefore, as used above, "resuspend" is incorrect and the term "suspended" should be used in all of the above cases since the original volume of the BSA is unknown.

Point number two is that The BCA assay is not a very good assay for proteins, I always use the Lowry method for proteins since it is specific for peptide bonds whereas the BCA reacts with lots of stuff. Good luck!

Appreciate it, thanks a lot, it's really much more clear to me now!

Bernard J Moncla : Smith's and Lowry's assay work by the same principle: Cu2+ is reduced to Cu+ by the peptide bond in an alkaline environment, the latter is then detected by BCA or molybdic acid, respectively. Thus, both assays are sensitive to reducing and complex forming agents.

My point was that the Lowry does not react with carbohydrates and the BCA does. Giving inaccurate values. That's all, switch to the Lowry and you will get more accurate results, especially if you are working in a gel system.

Bernard J Moncla : Actually, BCA is more resistant against, for example, sucrose than Lowry (http://tools.thermofisher.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf).