I have been using streptavidin as a constuct scaffold. Once solubilised in PBS, I have been having issues with fluctuations in the spectophotometer absorbance reading.
An additional issue is that the straight stock solution produces a much lower absorbance than the extinction coefficient provided would calculate to. But regardless of this, when reading, the absorbance does not show linear increase when the concentration is doubled (sometimes, it does not show an increase at all; there have even been occasions when the initial absorbance I have shown has disappeared when adding in more sample!)
Does anyone have any experience with this? Could it be that the streptavidin is sticking to my cuvette? I am using Quartz cuvettes, so i'm not sure how to overcome this issue.
Thanks!
Brittany
Not 100% sure. Have you tried altering the frequency of the reads? Could be a lower frequency might prove to give more accurate results. I'll try to be more helpful after work.
Laurence
UPDATE so, had a look and the only thing I can really think of is the high binding affinity could be causing for stv to bind to PSB and then break off, rebind in a cylce, this would cause a fluctuation in your readings. Again I could be completely wrong so would implore others to answer, and suggest trying a different machine :/
Thanks very much for replying Laurence, I have tried different frequencies and also different machines (I was initially using a very basic spectrophotometer and attributed the problem to that, but we've had two new models coming in on test and I've seen the same problem repeatedly with both). It's bizarre - to the point where I can have an initial reading of 0.019 (at a 1:1000 dilution), then when I INCREASE to 1:500, the absorbance will not only decrease, but actually show nothing at all. Messy stuff!
I was initially solubilising the streptavidin in PBS, but more recently I moved across to water; which also had the same problem. I'll keep looking into it, thanks again for your help! :)
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes