I use the Bradford method to quantify my protein with the Pierce BCA Kit assay. Particularly I use the microplate assay, 25 uL of a standard solution is mixed with 200 uL of a reagent, and after incubation at 37 C for 30 min, the absorbance at 562 nm is read. There are 9 standard points ranging from 0 - 2000 ug/mL (2 mg/mL).
I got a good equation which was y = 0.001x + 0.0551
R2 = 0.9944
I purified a recombinant protein with affinity chromatography, it's histidine-tagged. I kept all the last eluent.
What I want to ask is,
After concentrated my protein with an amicon centrifugal filter unit, I obtained at least 500 uL.
I readily quantified this solution with the same condition as above i.e. 25 uL aliquot was mixed with 200 uL of the reagent, left for 30 min at 37 C, and A562 was read.
Let s say the absorbance is 1.500, and with the equation I'll get:
1.500 = 0.001x + 0.0551
x= 1444.9 ug/mL or 1,44 mg/mL
Yes, I could use this concentration for my enzymatic assay. But what baffled me is....
How much the actual amount of the protein in my 500 uL ?
Because I want to incorporate this protein into a certain medium. And my teacher said to me, no need to lyophilize my protein. Just directly use it.
But How could I adjust the concentration of the protein? e.g. 0.01%, 0.025%, and 0.05%. The media is a powder. The protein needs to be mixed with the powder and then a homogenous dough is obtained (with the addition with ddH20).
Thank you
500 microliter is half a milliliter, therefore if the concentration is 1.44mg/ml, you have 0.7 mg. To mix in the lyophilized protein with the media powder would be a very unusual procedure.To lyophilize the solution almost always leads to some loss of the protein. Since 1ml = 1g of water, you have a 0.14% solution of your protein, which you can dilute to get the needed final concentration.
Annemarie Honegger Thank you so much for your suggestion. Yes, actually I need to do an in vivo assay to evaluate the effects of my protein on insects. It s called cystatin, a type of protein inhibitor. Hence, I need to mix it with a powder to make artificial seeds (feeds for the insect)
I want to ask another thing, I don’t know this is correct or not, because I think this one is such a nonsense.
My senior told me, that...
from my equation, after I got the “x” value, I need to divide it by 25, to obtain the protein concentration in ug/uL.
Thus, it should be 1444.9 divides by 25, equal to 57.796 ug/uL or 57.796 mg/mL for the total soluble protein.
Is it right, I couldn’t see the logic.
OK - you have done your standard curves: different known dilutions of your standard (concentration in mg/ml or ug/ml?), of which you add 25 ul to 200 ul of reagent. For your sample, you do the same: you also add 25ul to 200ul of reagent. Therefore from your standard curve (you checked that it is linear over the full range, i assume) and the absorption you measured for your sample, you can directly read off the concentration of your sample in the same concentration units you used for the standard - no need to correct for the dilution by the reagent, since this is exactly the same for the sample and the standard and therefore cancels out.
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Your standard curve tells you: for a concentration of a, you read an absorbance of b, and by interpolation on your curve, you can directly read off what concentration corresponds to the absorbance you measured for your sample.
Any dilution you did going from your stock solution to the 25 ul sample you used in your assay, you have to correct for: for example, if you diluted 1:100 before taking your sample for the assay, your stock solution is 100X more concentrated than the sample you used in the assay.
If you lyophilize such a dilute sample, it can easily happen that some of the very fluffy powder is aspirated by the pump and lost. In addition, it is difficult to mix two powders very uniformly. Since you mix your media powder with water, you dilute your Cystatin solution into the water you use to prepare your artificial seeds, and thereby get an even distribution without the need to lyophilize.
Ms. Annemarie Honegger your comment really clear my mind. The one that my senior told me was definitely wrong. I cultured my bacteria in 50 mL LB, induced with IPTG and incubate for only 3 h in 37. If I get a solution with total protein of 50 ug/ul from that culture, it is "unreal".
And also thank you for your helpful suggestions. I will try that too.
Best regards.
Minde is right: there is no perfect protein assay. A Bradford assay typically will give you a value that is within a factor of 1/2x to 2x of the real value. And if you always use the same assay, your results will be reproducible. The easiest way to know how much protein you REALLY have is to dialyze against water and then lyophilize and weigh it.
I am sure you got your answers from previous suggestions by other scientists. True there is no perfect protein assay. However, you may also try our improved membrane based protein assay. Thank you.
Heda GD, Kunwar U, Heda RP (2014). A modified protein assay from micrograpm to low nanogram levels in dilute samples. Anal Biochem 445C:67-72.
One thing you should keep in mind is that absorbance readings become the more unreliable the higher they are. The more light is absorbed by the sample, the less is received by the detector, and the more the result is influenced by noise. Most photometers can't read reliably beyond 1.0 (the exception are laser gel scanners).
In addition, due to inner filter effects, the absorbance vs concentration curve is linear only up to a certain value, then levels off. For the Bradford assay, this is an absorbance of 0.4 or so. Up to a degree, you can correct for that by using a quadratic standard curve (y = a + bx + cx2), but if you want reliable results, it is better to dilute your samples so the maximum absorbance is still in the linear range (but the minimal absorbance above the lower limit of quantitation).
One last point about the Bradford-assay: Because serum albumin has hydrophobic pockets for hormone binding, it binds more dye than other proteins, giving an anomalously high colour yield in that assay. It is therefore unsuitable as standard protein, use IgG instead.
From my experience:
All photometric assays that rely on a standard curve will always just give you a good estimate. Use a different standard (e.g. IgG vs BSA) and you get different values. Still ok for crude mixtures. For purified protein we compared amino acid anaylsis with OD280 based on the sequence-derived extinction coefficient (the theoretical one) and they were close enough to give me piece of mind that OD280 works well for most of our proteins.
Cheers
Peter
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