For an experiment i need the small cleavage products of Hyaluronan, meaning that i need especially the activity of Hyal-1 in the product H3884 of Sigma.
I guess this is why standard protocols use the sodium acetate buffer with a ph ~5
After the cleavage reaction i need to incubate with a protein which needs ph 7,6 for optimal results. Additionally this protein looses binding capacity below pH 6.
So the question is (or the questions are)
1. can i simply increase the ph after the cleavage reaction by adding NaOH
2. if it exceeds th buffering capacity can i simply use citric acid buffer for my cleavage reaction and increase pH then
3. is there still residual Hyaluronidase-1 activity at ph 7,6 so that i just incubate my mixture for a longer period of time?
As you can see. The problem i need to workaround is that i NEED pH+7 for the second reaction. Can i achieve that somehow without being in the need of uisng ion exchanging or other seperation methods?
I do not use complicated mixtures. The first mixture only consists of the sodium acetate buffer, hyaluronidase and purified hyaluronan.
Any help is much appreciated!
Thanks
@Monyer Al-Fatlawi: have you read an article or Review which mentions or even solves the problem. I'm in deep literatur research and haven't found anything yet. Any help is much appreciated.
Thanks
As no answer were posted, i went for the trial and error approach.
A little more to the issue:
For my experiment i needed a pre-incubation of a biotinylated protein with hyaluronic acid cleavage products in order to stain paraffin embedded tissue with alkaline phosphatase and permanent red.
The problem was that this pre-incubation set the pH of my mix to 6,0. Preliminary testing showed that the protein wouldnt bind under this conditions.
My approach was to create TBS with the pH 8,4
After adding this to my preincubation the pH of the mix was 7,4 which was the perfect ph for the binding ability of protein.
The results showed that the staining was rescued (in comparison to the staining at pH 6,0) but it still lost (marginal) amounts of staining intensity.
If this approach is applicable to cell culture, i will post the results here
So far it looks as this could be a decent approach.
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