Hello,
I'm purifying protein which lacks aromatic residues and contains very little hydrophobic residues. Thus, the only way to measure the protein concentration is BCA assay, as Bradford gives me completele unreasonable results.
At first I measured 2 Pools after SEC purification:
Pool1 C = 0.8 mg/mL
Pool2 C = 0.6 mg/mL
Then I mixed Pool1 and Pool2 in equal volume ratios and the concentration was 1.1 mg/mL.
But the most surprising thing was that when I measured concentration of both pools after -80C storage it was:
Pool1 C=1.9 mg/mL
Pool2 C=1.7mg/mL
Do you have any ideas what is going on? I am very confused because what I expected was the protein loss during freezing-storage-thawing, and this is something completly unusual for me.
Thanks for any hints
One reason could be protein degradation or structural changes. In BCA Cu2+ forms coordination bonds with some amino acid residues such as Cysteine, Tyrosine etc. Also Cu2+ can form strong coordination bonds with free -NH2 groups. If your protein degrades during storage, the amount of -NH2 groups increases. If there are disulfide bonds degredation might expose free -SH groups which may also increase bonding with Cu2+. In addition various structural changes during storage can change which amino acids are easily accessible by Cu2+ ions
It is important to note that all protein assays, be it Bradford or Lowry or BCA have their advantages and shortcomings. None is 100% perfect.
In this case, the freezing and thawing process could have led to the denaturation of your protein samples. The denaturation could have caused the release certain single amino acids such as cysteine or cystine, tyrosine and tryptophan which will also produce color and interfere in BCA assays. This could be responsible for the increase in the readings which was interpreted as increase in protein concentration.
Are you careful to remix the samples after freezing and thawing? When solutions are frozen slowly, the solutes, including proteins, can separate from the ice and concentrate at the bottom of the container. Protein solutions should always be frozen rapidly (using liquid nitrogen or dry ice), and remixed after thawing.
Hi Malwina,
During storage , you will have evaporation. This will, in turn, concentrate the protein solution and that can easily explain why your measurement are different pre and post freezing.
Losses occur at low concentrations due to sticking of the protein to tubes. Did you observe the volume of the sample to be different after freeze-thawing?
Best,
Hediye.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line