I am using superdex-75-HR-10/30 to separate two 23 and 7 Kda proteins. The protein concentration is 18µg/µl. It is a recombinant protein which have been cut using prescission protease. FPLC Buffer contains 0.15 mM NaCl, 0.05 Tris, 1mM DTT, pH: 8, and the maximum flow rate I can go to is about 0.40 ml/minute. It shows an absorbance for 23 kda protein after 16 min protein run ( 8 ml) but not for 7 Kda protein. Approximately after 30 minute protein run (15ml), conductivity falls down dramatically and in two minutes (1ml) it increases to its stable level again, but there is no significant absorbance here and thus no protein.
Can you help me please to purify my protein? do you know what has happened to the protein?
What are the results from running SDS-PAGE on the fractions? Do the 23 and 7 kDa proteins come out in the same fraction. If so, then the 2 proteins may be associating. Try using a higher concentration of NaCl, like 500 mM.
Thank you very much by the answer.
before running the protein in FPLC I checked the result of digestion, the protein had been digested completely. after FPLC, on SDS-PAGE gel there was only 23kDa lane in 8th ml and no other protein lane was seen on gel.
I would imagine your 7 kDa protein would elute in the void volume . Does it have a chromophore so it can be detected during the FPLC run? Could it be degraded by protease?
I have attached the chromatogram, the protein concentration was low in this run. after FPLC, I checked peaks on SDS-PAGE but non of them was 7kDa.
Based on the chromatogram you have two peaks labeled for the 23kDa protein. My guess is that the 7kDa protein is bound to the 23kDa and when you run your SDS-PAGE the 7kDa peptide/protein has not entered the gel or it's not staining well enough to be seen. Also it is possible it is covalently bound to to 23kDa protein and therefore does not dissociate from it and, depending on the gel being used, may appear to be 23kDa in size.
Have you looked in fractions B16-B6 for the 7kDa protein - or only in the peaks you have labelled? It looks like there is material coming off the column right up to the end of the run and your 7kDa protein should be in that region.
It looks like you are collecting fractions based on a minimum UV absorbance, I would alter your method to collect all eluate. If your 7kDa protein doesn't contain an aromatic residue it will not absorb UV, and consequently will be sent to waste instead of your fraction collector. What are you staining your SDS-PAGE gels with? Try using something more sensitive than coomassie, like silver stain or Oriole (Biorad).
thank you all.
following your suggestions I used 500mM NaCl instead of 150 mM, and checked all fractions but with coomassie yet. the polypeptide has 4 aromatic residues. I concentrate the polypeptide solution before injection to FPLC and second peak disappeared which seems belonged to DTT. but there was not any 7Kda polypeptides in any of fractions again.
- Badges
- Science method