Generally nucleic acids have a peak absorbance at 260nm

Alanine residues absorb at 260, maybe the lack of Tyr and Trp residues causes the shift. But the 280 maximum of previous preparations may indicate a diferent product of expression, maybe a shorter amino acid chain? No postraductional modifications are possible in bacterial expression. A ligand or contaminant with strong absorption at 260 nm could also be the cause, as Yazad mentioned above.

Hi there,

It sounds like nucleic acid contamination, as it's a basic protein it might interact non specifically with DNA/RNA. The question is why this contamination hasn't occured in the previous experiments? Try to spot what has been changed in between.

Another point : why are you working at pH9.3 which is very close to the theoritical pI of the protein? This is not indicated from the protein stability point of view.

To get rid of nucleic acids, you may pass your protein sample onto heparin column with salt gradient (make sure the protein is actually positively charged so that it will interact with the matrix).

As previous fellows said it looks like DNA contamination. I faced this problem before and solved this by using higher concentration of NaCl (100mM to 300mM) in the washing buffer.

You can also get rid of DNA by anion exhange, as your protein is basic and is not going to bind it, you can use a Q-FF as a negative capture step for example to eliminate DNA and a large % of E. coli proteins which are mostly acidic proteins. At the same time you will save your gel filtration column cleaning up the clarified extract previously to load this column which is most useful for polishing steps.

Of course, be careful with elimination of DNA by other methods because sometimes your protein could go with DNA because the complex is too strong. For example, PEI will eliminate DNA by centrifugation but the proteins which bind to the DNA normally goes with PEI also. Always check by SDS-PAGE.

Good luck

I wan´t to thank all your responses which will help us a lot to solve our problem.

Best regards,

Rafael

The presence of nonprotein chromophores and nuclei acids can increase Abs260. If nuclei acids are present the following formulae can you help to determinate the protein concentration

Protein (mg/mL) = 1.55Abs280 - 0.76Abs260

Protein (mg/mL) = (Abs235 - Abs280)/2.51

Protein (mg/mL) = 0.183Abs230 - 0.0758Abs260

Layne, E. (1957) Spectrophotornetric and turbidimetric methods for measuring proteins.Meth. Enzymol. 3, 447–454.

Whitaker, J. R. and Granum, P. E. (1980) An absolute method for protein determinationbased on difference in absorbance at 235 and 280 nm. Analyt. Biochem. 109, 156–159.

Kalb, V. F. and Bernlohr, R. W. (1977). A new spectrophotometric assay for protein in cellextracts. Analyt. Biochem. 82, 362–371.

It might be caused by nucleic acid contamination. You could remove it by further purify your protein with heparin column or Q column (heparin column will be better here). You can also add some DNase and RNase when you lyse the cell, and use higher salt concentration in the lysing buffer and washing buffer (300-500mM) .