Hello all,
When performing SDS-PAGE electrophoresis under non-reducing conditions (without BME and DTT), disulfide bonds were broken. I am using a mPAGE 4-12% Bis-Tris Gel and MES buffer for the analysis. The denaturation process of samples was performed in a thermocycler device at a temperature of 95 degrees.
What could be the reason for protein reduction?
Dear Natalia
how long did you boil at 95°C your samples?
Because for example for mabs i found that 5 minutes at 95°C is too long and i saw several degradation bands that disappear if i reduce the incubation time at 1' and this is much more in agreement with the SEC profile of our mabs that do not show any main degradation at low MW in analitic SEC.
Is you protein contain free cysteines?
Because it seems that disulfide-bond scrambling could be catalyzed by free sulfhydryl groups and in case you can prevent it by adding a cysteine blocking agent as iodoacetamide or similar.
give a look to the following paper
https://www.sciencedirect.com/science/article/abs/pii/S0731708513001842?via%3Dihub
Ciao
Manuele
Dear all, the following documents deal with the disulfide bond chemistry. My Regards
https://doi.org/10.1002/cbic.202000092
https://doi.org/10.1146/annurev.micro.51.1.179
https://doi.org/10.1080/1040841X.2018.1538933
David Farringdon Spencer
Hello: I am also performing non-reducing SDS-PAGE. Can long exposure to heat disrupt the disulfide bond? I boil my samples for 98 oC for 10 mins (no B-me) and I observe that my dimer band (the one with SS bond) is really thin and faint.
Disulfide bonds can be cleaved at an alkaline pH by treating a protein with excess of a reagent disulfide in the presence of catalytic amounts of thiol. The cleavage products are stable and can be isolated; they contain the mixed disulfide between the reagent and the exposed thiol groups of the protein. https://pubmed.ncbi.nlm.nih.gov/5866656/
Check "The susceptibility of disulfide bonds towards radiation damage..." https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467163/
Hi Dr Natalia Urbańska . I agree with Dr Abdelkader BOUAZIZ completely.
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