Hello everyone,
I am trying to see if my protein has irreversible, reversible, or it forms disulfide bonds. Previously, I mixed my protein ( 25uM) with MAL-PEG 5000 ( 250 uM) to see if the maleimide will react with the protein, It did not react with the protein, so the molecular weight did not go up. This time i want to reduce the protein to see if the cysteines can become free or to see if the cysteine are buried within the protein. I know tcep is a better reducing agent than dtt because tcep can react without being in competition with the maleimide. the buffer that my protein is in is sodium phosphate p.H 7.4. My question is how much tcep do i add ( morality) and how long and what temperature does it need to incubate and to know if the cysteine have been reduced. p.s my protein has 2 cysteine.
Hi Jesus.
This is a bit of a late reply but in case you are still working on that project, I conjugated PEG Maleimide to several proteins using previous TCEP reduction at 37°C for one hour. Then proceed to Ellman's colorimetric assay is useful to calculate molarity of free thiol groups which you obtained so you can adjust 10 to 20 equivalents of your PEG.
Hope this will help, if not you at least another reader maybe !
All the best !
@Francoise Bevillard can you please share protocol for Ellman's colorimetric assay to calculate molarity of free thiol groups. I need that
I reduced by antibodies using TCEP. After that I wanted to measure the amount of free thiol groups but when I use Ellman reagent, I got a reading even for the control tube with has TCEP but no antibodies. Does TCEP interfere with Ellman reagent?
TCEP is not compatible with Ellman's reagent. See reference below
http://www.ncbi.nlm.nih.gov/pubmed/7978256
TCEP also breaks DTNB to give NTB ions.
TCEP is not compatible with Ellman's reagent. See reference below http://www.ncbi.nlm.nih.gov/pubmed/7978256
@ Francoise Bevillard did you obtain good yields? I've been tried conjugating proteins with PEG-maleimide using TCEP as reducing agent and it completely failed. Indeed, I found in the bibliography that PEG interferes somehow with TCEP. Could be a matter of TCEP concentration? Which should be the optimal ratio protein:TCEP:PEG-maleimide?
Thank you!
Marta Camps hello, did you make some progress with the conjugation of proteins to PEG-maleimide? What amount of TCEP did you use with respect to the protein?
Hi Cynthia Bessem Nkemeto , sorry for the delay in may response.
I keep struggling with TCEP concentrations and protein (2 cysteines) conjugation with PEG-Maleimide. I performed the reaction at 10, 20 and 200µM but even at 10µM TCEP my protein completely lost its reactivity. So finally I removed TCEP from equation and my protein could be conjugated while keeping its reactivity. Herein, I don't know if PEG is somehow playing a role in this loss of reactivity...
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