two proteins(23 kDa, 66 kDa) shown same band intensity on sds page with coomasie stainining. Then i measured the concentrations of both proteins with nano drop at 280nm directly, 1. with out considering ext. coefficient, MW and 2. with considering ext. coefficient, MW of the proteins... 1. measurements given concentrations like (0.5 mg/ml, 0.01 mg/ml); 2. measurements given concentrations like (1 mg/ml, 0.1 mg/ml); both measurements have given different values...now can anyone suggest me which measurement need to consider?
Hi Babu,
The fact that you get closer values when compare coomassie staining with A280 when you don't consider extinction coefficient is a coincidence. If the value you get using a 'generic' ext coef is significantly different from when you use the correct ext coef then this is a measure of how wrong your 'generic' ext coef is. If your concentration using A280 with the correct ext coef is significantly different from the actual value, then this is a measure of the purity of your sample.
Alok, using densitometry is simply another way of measuring the binding of your protein to coomassie. If the value you get here is significantly different from the 'actual' concentration value then this is simply a measure of the difference in coomassie binding between your protein and the chosen standard. Using this method is worthwhile once you know that there is no significant difference in coomassie binding between the test protein and the standard - unfortunately we seldom know this a priori.
One thing is certain however, using the wrong ext coef so that you can better match one concentration to another is simply making things up. The only time there is any justification to use a 'generic' ext coef is when you don't know your protein sequence - and in this case you should still be very aware that the difference in concentration between a Trp rich protein and one lacking Trp residues all together can be more than an order of magnitude.
Andy
Hi Babu,
There are a number of things which you need to consider here.
Firstly dye based methods assume that your unknown protein sample binds to the dye at the same rate as the standard you use for calibration. This can often be the case, but I have personally had many cases where the difference in dye binding for the unknown and the standard vary by more than an order of magnitude.
Secondly, absorbance based methods assume that the sample you are using is very high purity and contains no contaminating molecules which absorb at the wavelength(s) you're using for your measurement. Only you can be the judge of the purity of your protein samples, but you need to be aware that your calculated concentrations can be wildly different from the actual concentration when your sample is contaminated with even small amounts of something that absorbs strongly at the particular wavelength you're using. Moreover, the contaminating species may not necessarily be a protein and could be a small molecule for example, and it is usually worth recording an abosrbance spectrum to check that the profile looks reasonable for a protein.
Finally, if you are using A280 then you need to know the extinction coefficient for the protein you wish to quantify - this is highly dependent on the amino acid composition of your protein and in particular the relative frequency of Trp residues. I have worked with several proteins that don't contain a single Trp residue and these proteins therefore have a very low extinction coefficient. Other proteins I've worked on have quite a high relative frequency of Trp residues and these proteins therefore have a high extinction coefficient.
So to summarise, the dye binding and abosrbance based methods each have their uses provided you understand their limitations, and never use A280 without taking the extinction coefficient into consideration.
Let me know if you need clarification of anything in the above.
Andy
Hi Andrew
Thank you for the reply. But the values based on band intensity by coomasie and protein concentrations measurements with out extinction coefficient were almost matched but the value which i got by with ext coff given more differnce with coomasie band....now I am confused which i need to consider?
Best would be to do a densiometric analysis. Run different concentration of a known (any standard protein) along with different volume (say 2, 4, 6, and 8 uL) of your protein on SDS-PAGE and compare the amount.
Measure 280 OD in a traditional spectrophotometer, then use the ext coef for concentration calculations. (More later).
Hi Babu,
The fact that you get closer values when compare coomassie staining with A280 when you don't consider extinction coefficient is a coincidence. If the value you get using a 'generic' ext coef is significantly different from when you use the correct ext coef then this is a measure of how wrong your 'generic' ext coef is. If your concentration using A280 with the correct ext coef is significantly different from the actual value, then this is a measure of the purity of your sample.
Alok, using densitometry is simply another way of measuring the binding of your protein to coomassie. If the value you get here is significantly different from the 'actual' concentration value then this is simply a measure of the difference in coomassie binding between your protein and the chosen standard. Using this method is worthwhile once you know that there is no significant difference in coomassie binding between the test protein and the standard - unfortunately we seldom know this a priori.
One thing is certain however, using the wrong ext coef so that you can better match one concentration to another is simply making things up. The only time there is any justification to use a 'generic' ext coef is when you don't know your protein sequence - and in this case you should still be very aware that the difference in concentration between a Trp rich protein and one lacking Trp residues all together can be more than an order of magnitude.
Andy
Best would be to do a densiometric analysis. The fact that you get closer values when compare coomassie staining with A280 when you don't consider extinction coefficient is a coincidence. If the value you get using a 'generic' ext coef is significantly different from when you use the correct ext coef then this is a measure of how wrong your 'generic' ext coef is. If your concentration using A280 with the correct ext coef is significantly different from the actual value, then this is a measure of the purity of your sample
There are a number of things which you need to consider here
Firstly dye based methods assume that your unknown protein sample binds to the dye at the same rate as the standard you use for calibration. Secondly, absorbance based methods assume that the sample you are using is very high purity and contains no contaminating molecules which absorb at the wavelength(s) you're using for your measuremen. Finally, if you are using A280 then you need to know the extinction coefficient for the protein you wish to quantify - this is highly dependent on the amino acid composition of your protein and in particular the relative frequency of Trp residues.
Hi there,
Band intensity is dependent on the protein ability to bind Coomassie which may vary a lot from protein to protein so this is not a reliable estimation unless you have the specific calibration making the link beween intensity of the band and the actual quantity of protein loaded.
In the lab we always rely on the calculation from Nanodrop data using the specific coefficient calculated from protein primary sequence rather than gel coloration. Gel coloration is only used for estimating protein sample purity.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins