Hi,
I will be quantifying the protein-protein interaction using the β-Galactosidase liquid culture assay with ONPG as substrate. I am following the Clonetech's yeast protocols handbook (PT3024-1). In this protocol, it mentioned as
"16. After the yellow color develops, add 0.4 ml of 1 M Na2CO3 to the reaction and blank tubes.
Record elapsed time in minutes.
Notes:
• The time needed will vary (3–15 min for the single-plasmid, β-gal-positive control; ~30 min for a two-hybrid positive control; and up to 24 hr for weaker interactions).
• The yellow color is not stable and will become more intense with time. You will need to run a new blank tube with every batch.
My question is when to add the 0.4 ml of 1 M Na2CO3 into the samples? How many samples we can manage at a time? I have about 15 interactions to test.
Any guidance will be greatly appreciated.
Thanks,
Ragu
Hello, when yellow color develops it means in ONPG structure the bands between Betta D Galattose and O-Nitrophenol disrupted. Yellow color from O-Nitrophenol which is more acidic than phenol itself and the addition of Na2CO3 needed for neutralization and pH regulation. So you need to add Na2CO3 after yellow color formation. How many samples can you perform? depends on your capabilities and performance.
Good Luck
I would strongly advise you to first do a kinetic reaction function of time then you can decide after how long you should stop the reaction. This is a classical way how to proceed in enzymology.
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