I am using the following reaction condition
Thermofisher trypsin dissolved in 50mM acetic acid (1mg/ml): 2 uL
Protein sample: 50 ug (dissolved in 50mM ammonium bicarbonate, pH 8)
Make the total voulme : 50 uL by adding 50mM ammonium bicarbonate, pH 8 in the reaction mixture
Total protein conc. 1 mg/ml
After trypsin digestion and adding formic acid upto 1% and centrifuging in cold temperature (4 degree). I found the precipitate and check the protein content of the supernatant around 0.029 mg/ml.
Even MS/MS analysis does not look good. What should I do improve the sample preparation ??
Do you quantify the amount of protein you are using before you start the digest? You might have a low volume of protein before the digest.
I am suspecting that the problem is with the acetic acid used in high concentration for suspension of trypsin that might have hinder the digestion of protein . And the protein conc. before and after acidifications shows difference.
before acidification : 1.2 mg/ml
after acidification: 0.029 mg/ml.
thanks for your time rachel
Hi Arun,
I think the initial concentration of acetic acid that you used was too high. Remind this fact that trypsin is only functional at the pH values between 7-9 (37 degree Centigrade). I'd repeat the sample prep using reasonable pH values in this case. For the chromatography, check the final pH of the sample before your injection as well. Too acidic samples (even the injection volumes are very low) would result in poor chromatographic separations.
Hope you can get better results.
Regards,
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