Hi all, i hope someone can help.

I am having a lot of problems using the Micro BCA assay to determine the concentration of my protein, and hope someone can give me some advice.

I have purified my protein, and in the final steps of the purification it was dialysed against water for three days (5L at a time), so it should be pretty clean. I added water to the dried protein, aliquoted into equal amounts, freeze dried, and stored in the freezer.
I then resuspended an aliquot in 100ul water to try and do a micro BCA assay.

The standard curve with the BSA standards they supply always comes out perfectly fine, whether i try the test tube or the microplate procedure. I generally do three different dilutions of my protein, so i will use 1ul/ml, 5ul/ml and 10ul/ml (for the test tube procedure). The values i get NEVER correspond to each other - I mean that 10ul is nowhere near ten times the 1ul, which is never 5 times less than the 5ul. None of the values correspond to the nano drop reading either, the last time i did this, 1ul for the BCA assay gave a reading of 11ug, 5= 19.5ug and 10ul = 29ug.

What is going on? Is it possible that the range of the micro BCA assay is too small for the concentrations i am using, so i am just getting inaccuarate readings for those which are at the extremes of the curve?
Could my protein be aggregating etc in the water, and if so, which other solvents would be best to try? I don't think it is pipetting error as the standards come out perfectly fine each time.

I have tried this about ten times now, and never had a decent result. I should also mention that i replicate each dilution at least twice, and there is no problem here. The readings are all very much the same.

Thanks in advance for any help!

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Oh and also, after reading some of the other posts i have a quick question about drawing the graph - I have up to now been using sigma plot to draw a linear line for my standards - is this correct?

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if you are at the extremes then you may be inaccurate. what are the actual readings? you want them to be between 0.1 and 0.7 (or 1.0, depending on your spectrophotometer) for maximum accuracy.

have you tried the standard bca assay? maybe that range will be better for your protein.

at least it is reproducible. this leads me to wonder if your standard curve is truly linear.

or

how accurate are your pipettes? are they calibrated? did you use the same pipette for all three volumes? you may want to check their calibration.


only if your data is linear. if not then you should fit the curve and evaluate your readings from that.

if your protein concentration is out of the range of the assay then you need to dilute or use an assay with a more appropriate range.

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hello,

The assay kit provides a BSA standard, which is made up at the following comcemtrations - 40ug/ml, 20, 10, 5, 2, 1, 0.5 (200ug/ml is also run, but this readimg is much too high for the spectromphotometer. the kit is supposed to read concentrations between 2ug and 40ug/ml.

A typical reading for the 40ug is ~1.7 and for 0.5ug about 0.05 (blank corrected). After actually reading tech tips from the company, i constructed a graph using a polynomial trendline, and worked out the concentrations of my unknown samples from the equation, as suggested.

The readings for my protein samples (blank corrected) and therefore the concentrations according to the equation are as follows -

1ul 0.1495 = 2.87ug/ul
2ul 0.401 = 8.92ug/ml
6ul 0.958 = 22.65ug/ml

(so really these concentrations are per ul as i have diluted them in 1 ml of water for the protocol)

So these concentrations should be well within the supposed range of the assay? i suppose the 2ul and 6ul are not too far off this time... This time round i used pipettes which had just been calibrated, and yes i use the same pipette for each of the three concentrations.

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it looks to me that you took the reading from the curve and just called that the concentration without taking into account the volume of the protein solution that you added to the assay (diluted to 1ml). if you divide the 2ul by 2 and the 6ul by 6 then you get 4.46 and 3.775ug/ml. these values match up better, although not perfectly. the average of all three comes to 3.702ug/ml. these values are by direct reading, i assume, but you diluted the samples. if you apply the dilution factors to the results then you will have an average of 3.702mg/ml.

you may find this publication by pierce to be enlightening:

read standard curves

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