Hello
Different molecular weights proteins 23 kDa and 66 kDa looks similar band intensity on SDS-PAGE(coomasie staining) does it mean both proteins contain same concentration? When I measured these protein concentrations with nano drop its shows lower MW protein was 1 g/ml and higher MW protein band was 0.2 g/ml....why is it showing different concentrations with nano drop? am i wrong somewhere?
Thank you
One possibility is that they bind CBB in different ratios. Proteins with sufficiently different aminoacid compositions (especially basic and hydrophobic aminoacids) bind different amounts of CBB per mass unit; this is one of the shortcomings of the Bradford assay.
Another possibility is that they have very different absortion coefficients. If you used the same coefficient for both in the nanodrop measurement, this might be throwing you off.
Also take into account that even if they bind CBB at the same ratios, equal intensities will mean equal mass, but their molar concentrations will be different -one will be approximately one third lower than the other.
Proteins interact differently with protein stains, staining intensity of different proteins at identical protein loads can be very different. Even it looks same quantity but the concentration will be different.
Same band intensity means their amounts loaded are same on the gel (it is a measure of absolute amount and not concentration), assuming they interact with commassie in a similar way. In that case, what you have written makes sense- lower sized protein has higher concentration in your sample and this is inverse for the higher sized protein. Here I am making another assumption that both bands are observed in the same lane, meaning loaded amounts were same for both proteins.
Protein with bigger size always stains more than lower size. So if your lower size protein is of similar intensity as of the other, it makes perfect sense that their staining is of the similar intensity.
You can measure band intensity to get a ratio of your bands by using the free online software from NIH (ImageJ).
The intensity of the band does not mean that high concentration of the protein was obtained, so we have to use Nano drop to measure the concentration of the protein.
I would like to point out that your concentration readings seem absurdly high, 200mg/mL of a 46kDa protein? And higher for the other?
@Alejandro Martin
One possibility is that they bind CBB in different ratios. Proteins with sufficiently different aminoacid compositions (especially basic and hydrophobic aminoacids) bind different amounts of CBB per mass unit; this is one of the shortcomings of the Bradford assay.
Another possibility is that they have very different absortion coefficients. If you used the same coefficient for both in the nanodrop measurement, this might be throwing you off.
Also take into account that even if they bind CBB at the same ratios, equal intensities will mean equal mass, but their molar concentrations will be different -one will be approximately one third lower than the other
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Thank you for the reply Martin...
I didnt use coefficients at all to measure the concentration of them. I measured the protein concentration directly with nano drop at 280 nm.
I have one more query that, how much we can trust these protein concentrations measured at 280 nm?
@ Nitin Sabherwal
Same band intensity means their amounts loaded are same on the gel (it is a measure of absolute amount and not concentration), assuming they interact with commassie in a similar way. In that case, what you have written makes sense- lower sized protein has higher concentration in your sample and this is inverse for the higher sized protein. Here I am making another assumption that both bands are observed in the same lane, meaning loaded amounts were same for both proteins.
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Thank you for the reply Nitin
I didnt load both of them on the same lane but different lanes to see to same intensities
@Daniel Hall
I would like to point out that your concentration readings seem absurdly high, 200mg/mL of a 46kDa protein? And higher for the other?
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Thank you for the reply Daniel
how it is absurd? it suppose to be low concentrations I get?
Daniel is right, you _can_ concentrate some proteins to 200-400 mg/mL but the solution becomes a paste. There is something wrong there.
As for your question re: coefficients yes, relying on absorbance at 280 nm in a nanodrop can only be used as a rough guide, as the actual absorption coefficient depends first on the number of Trp residues (and to a lesser extent, Tyr and Phe) and second, on the local environment of the absorbing residues. It is better to take coefficients from something like the ProtParam tool at ExPaSy (http://web.expasy.org/protparam/). This is still an approximation, but is appropriate for most needs.
Hope it helps!
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