I'm planning to conduct APEGS assay for the first time.
I got a protocol for the experiment, but have no idea how to prepare solutions used for APEGS assay.
Materials using for the assay are not familiar to me and seem to be very sensitive and fragile.
For example, methoxy-PEG-(CH2)3NHCO(CH2)2-maleimid (mPEG) is not stable in neutral pH.
Can I put mPEG in buffer (pH6) and freeze stock it (for 1 yr)?
I would like to know how others prepare and keep materials/stock solutions listed below in Lab?
N-ethylmalemide (NEM)
Tris(2-carboxyethl) phosphine-hydorchloride (TCEP)
Methoy-PEG-(CH2)3NHCO(CH2)x-maleimide (mPEG)
Hydroxylamine (HAM)
And for each experiment, how much (mL) do you need to prepare?
Thank you for your time!
Hi Fujiwara,
I am doing the APE assay. In my understanding, most of the reagents have to be fresh especially mPEG-Mal, NEM, HAM,TCEP. Not sure how your experiment goes so far. My calnexin control has a strong base band (no mass shift) and weak shifted bands (two bands shifted). Do you meet the same problem?
Hi Dr. Liu,
Thank you very much for your answer. I have not started this experiment yet, but I will prepare fresh mPE-Mal, NEM, HAM and TCEP when I start the experiment. Thank you for the practical advice.
Hello Masakazu,
I have been doing APEGS assay for more than 2 years now and characterised it as my protein of interest.
My experience:
The best way to do diluting whole bottle of mPEG (I use mPEG-MAL-10k) in dH2O, make aliquots, keep the aliquots at -80 until you use it. Otherwise, since it is very unstable, it is very hard to weigh mPEG every time before the experiment and you will also waste quite a lot of it while trying to weigh.
NEM has to be fresh, so you need to measure and dilute NEM in ethanol, every time before the experiment. Whenever I need to use it, I take some from the stock bottle which is kept in the freezer (-20).
Tris(2-carboxyethl) phosphine-hydorchloride (TCEP), Hydroxylamine (HAM) these two can be stored in the room temperature for ages, no need to aliquot it. I directly take some from the bottle with a pipette every time I use it.
I use 50% HAM solution.
I would say methanol-chloroform precipitation and right incubation times are the most important steps for APEGs assay. And every protein acts in a different way so it might take time to characterise the assay for your protein of interest.
Busra P. Yucel Thank you very much for showing your precise preparation methods. These tips are very helpful for the people who just started the new experiment like me. I really appreciate your help and kindness. Thank you very much.
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