Now I am using this kit to measure PDH activity in prostate cancer cell line PC3.
The NADH standard curve looks great.
The curve of cell sample in linear dilution looks great, too.
However, the parallel background control well (everything is the same except without PDH substrate) is MUCH HIGHER than its counterpart.
And this only happen in cell lysate samples. The positive control well/positive control without PDH substrate pair look normal: positive control well has higher OD value than its background control well.
Anyone knows about what is happening here?
Thank you!
Heng
Heng,
Essentially all cell lysate will contain pyruvate...the substrate for PDH...therefore you will have a high background due to the pyruvate being decarboxylated by the PDH.
Are you starting your assay with addition of pyruvate or addition lysate?
Do you account for the difference in volume for your substrate free reaction? Say you add 20 uL of substrate in a normal reaction, do you add 20 uL of H20 in your "substrate-free" reaction to account for the differences in concentration?
Normal reaction for cell lysate:
A: 20ul cell lysate+30ul PDH assay buffer
+
B:2ul substrate+2ul developer+46 ul PDH assay buffer
Background (substrate-free) well:
A: 20ul cell lysate+30ul PDH assay buffer
+
B:2ul developer+48 ul PDH assay buffer
Background (substrate-free) well is higher than normal reaction.
However, for Positive control well, results look normal: Background (substrate-free) well is colorless.
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