non-reductive gel to check the disulfide bond - (Aug/04/2006 )

Hi, guys,

I am trying to use non-reductive gel to check if disulfide bond were formed. Two residues in GABAR (one in alpha subunit, and the other in beta subunit, and they are very close in 3-D structure) were mutated to cysteine, GABAR was expressed in 293T cells and non-reductive gel (no ME or DTT) was used to check if there is disulfide bond formed between them (if there is, then a dimer appears when immunobloting one subunit with western. My problem is: I got a beautiful dimer band in the double mutated sample, but adding ME or DTT cannot break the disulfide bond (still a dimer, thought weaker than non-reductive gel). What am I doing wrong?

Bigworm

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How much ME or DTT are you using? How long do you denature? What pH are you at (esp. important for DTT, as it dies pretty quickly at alkaline pH)?

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ME-0.5%, DTT-50 mM, 40 min RT denature, pH 6.8

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Another finding: the dimer band disappears after simple boiling (without DTT or ME); Can disulfide bond break upon boiling (without reductive agent)????

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no. it should not break so easily.

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hi,

i have experimented another phenomenum when i ran a recombinant protein coupled to mouse Ig Fc domain on a SDS-PAGE in 3 different conditions:
1) w/ bME and w/ boiling
2) w/ bME and w/o boiling
3) w/o bME and w/ boiling

dimers appeared only in conditions 2) and 3), but the dimer:mono ratio was 9:1 in sample 3) and 1:1 in sample 2)...

Seb

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You mean disulfide bond does break upon boiling, just not as effective as using bME?

But my protein is membrane protein, most of them get aggregated upon boiling, that is why I dont' want boiling....

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Even if boil in the presence of SDS? My two cysteines are located in the transmembrane region....

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I mean: does SDS break disulfide bond when boiling? Not at all?

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It should not be as a chemical reaction, unless it was subjected to long oxidative envirument. I assume you only briefly treated samples, right?

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