I'm using a 30% polyacrylamide gel. I'm making a 6% gel with varied % concentrations of gelatin and varied concentration of trypsin.
For my prelinimary experiment:
6% gel
0.1% gelatin
0.1% trypsin
Note: I added trypsin ( trypsin powder was added DMSO to make the enzyme solution) after polyacryamide has gelled.
I cannot see any evidence of the trypsin digesting the gelatin. the experiment was ran at 20*C but i did not incubate the gel or trypsin.
What is the ideal condition to run this experiment?
is the gel concentration (6%) to high? the reason i'm using 6% is that i want a stiff gel. are there any alternative stiff gels?
I think two parameters are important to consider if we work with enzymes: temperature and pH. I worked in immunocytochemistry some time ago, and I used a trypsin solution to unmask cell epitopes. The concentration was also 0.1%, the working temperature was 37ºC (the optimal temperature for this enzyme) and the pH was set to 7.8.
Hi Karthik, you will have to walk me through your experiment because I don't understand.
You are using a 30% PAGE, and you are also using a 6% gel?
Are you describing an overlay assay in which you run a trypsin inhibitor on the 30% PAGE and overlay that gel onto a 6% gel that has copolymerized trypsin and gelatin?
It seems to be an interesting study. Not sure of the interaction chemistry between acrylamide and gelatin during gelation. Does it involve covalent interactions? Gelatin forms thermo-reversible gels, returning to viscous liquid at higher temperatures. You could raise the temperature to dislodge the gelled gelatin for digestion. Of course this has to be within limits to get optimum trypsin activity. As Jose Silva pointed out, a good starting set of conditions could be closer to to the optimum conditions for trypsin activity. Do keep us posted about the outcome of your experiments if possible. Good Luck!
Hi,
Thanks everyone for your answers.
Sarah, I actually used DPBS (without Mg and Ca) instead of DMSO to make trypsin solution.
I have CaCl powder, can i make 5mM concentration of this with 2O and add this to DPBS before i add trypsin powder?
At this moment (this is a prelinimary experiement to find the ideal gelatin and trypsin concentrations) i'm only visually checking if gelatin has been digesetd by trypsin. i'm assuming there will be holes formed on the PAGE gel.
However in the longer run i need to find a suitable method to check for digestion and inhibit trypsin - any recomendation?
Finally i'm using a stiff PAGE gel (6%) would this be a problem? thinking about the diffusion of trypsin through the PAGE gel.
Thanks for your time.
You could consider using dye quenched gelatin.
https://www.lifetechnologies.com/order/catalog/product/D12054
Thanks for your suggestion.
how can you make fluorescence labelled gelatin in the lab?
Can you attach UV markers to gelatin, so the products can be seen with a handheld UV light scanner?
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