We expressed intrinsically disordered protein following the protocol which we always used for this protein: expression in E.coli, destroying cells, adding streptomycin sulfate to get rid of DNA, salting out the protein with ammonium sulfate, resuspending the pellet in 10 mM Tris-HCl, pH 7.4, and boiling for 15 min. Resulting solution has super high absorbance at 260 nm, although this protein absorbs at 275. There is no DNA (nothing seen on the agarose gel). Afterwards protein solution was dialysed (didn't help), and then we did anion exchange chromatography (Resourse Q column). Our protein comes in one peak, as expected, but still absorption is too high, that suggests impurity, so we cannot use it. We tried to reinject fractions containing our protein after first chromatography second time, but it also didn't help, there is still very strong absorption at 260 nm. Does anyone know what it can be and how to get rid of it?
Not only DNA, RNA also gives high absorbance values at 260nm. If dna or rna is bound very tightly then it would be problem for your prep. You may try adding benzonase in your prep, and later you can try adding polyethyleneimine ( 0.1 - 0.2 % - recheck the final conc ) incubate on ice for 10 min and then centrifuge again
Dear kseniia
first of all..two stupid questions are you sure that the maximun absorbance of your protein is 275? how many trp, phe and tyr it contain? just because i my past i worked with some proteins as eg humam cu,zn sod1 that do not contain tryptophan and it is maximun of absorbance was determined from the phe present in the sequence
2) why are you boiling the sample. it seems to me quite strong lysis to be performed before protein purification.
however if the maximun absorbance is 275, i suspect that yoy have dna that is not removed from Streptomicin and that bind to che q coloumn with the protein. dna is negatevilly charged and bind well anionic exchange. (many dna purification coloumns are based on anoinic exchange)
in this case you can: Try to add some dnase or benzoase with a short incubation before the q coloumn and if after the elution you still have strong absorbance at 260 try to wash the sample by ultrafiltration with an amicon ultra or similar performing 2 or 3 concentration dilution step . in my experience dna treated with dnase or benzoase is fragmented and it will pass through the membrane (cut off 10kda or higher) and you will remove dna from the protein. if this approach work measuring the absorbance of the buffer that pass the menbrane you will detect signal at 260 but not coloratiin at bradford while the retained protein will provide blue coloration at bradford but decrased a260 and shift of the peak in the direction of 275nm.
best regards
Manuele
Dear Mahesh Chandak and Manuele Martinelli, thanks for your answers. I also thought first that it must be DNA, but when we ran the sample on the agarose gel, we did not see any signal at all. So it must be not DNA, but something else.
Also, sometimes we have very minor impurities of DNA in such samples, and then they are separated well by the chromatography. This time this strange impurity stays with the protein and comes in the same peak as the protein.
Concerning questions of Manuele:
1. protein is alpha-synuclein, and all the previous times, when we did not have this strange impurity, maximum of absorbance was always at 275 (it is not the first time we are preparing this protein, but the first time we have so unusually high absorbance at 260).
2. boiling helps to get rid of other proteins - all the folded proteins denaturate, and we centrifuge them out. Our protein is intrinsically disordered and it stays in solution. According to the gel, protein solution indeed becomes more pure, and then it is easier to perform final purification by chromatography.
The chain-chain interactions between hydrophobically collapsed protein molecules can promote intermolecular association resulting in the formation of higher order oligomeric species. As you are boiling and working with IDP such oligomer may form, can behave funny and might show high absorbance at smaller wavelength due to rayleigh light scattering phenomenon. Other than that DNA contamination, and impure ammonium sulphate issues are ruled out from your side.
I still think it is better to if you supplement benzonase along with your buffer.
I had a similar problem that turned out to be DTT. But you dont appear to have any of that in your buffer!
Hope you find the answer soon: I'm v. interested to know.
Normally in UV region we measure absorbance of protein at 280nm and for DNA and RNA we do it at 260nm . gettind no band of DNA in Agarose gel electrophoresis also may be due to less of DNA being loaded for run. CConcentrate and then check bands of Nucleic acid on Agarose gel.
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