I used on my protein extract from a tissue a high concentration of DTT(1M), I faced a problem because of this high concentration in protein quantification because all the methods have interfered with such high concentration of DTT. Is there any serious effect of this high DTT concentration on my protein separation on SDS-PAGE?and how can I quantify my protein?(I was thinking to dilute the sample but the problem I expect low protein concentration in my original sample which increase the problem in more dilution.
DTT will reduce the disulphide bonds of protein, so it might be interfering with the protein.
Eric: I have never seen any protocol using such a high concentration of DTT (1M), General methods use less than 2-10 mM, which are already higher than any internal (cellular) level. Could you explain more about the reason(s) of such a selection?
@Ke-Wei Zhao: I did a mistake in calculation so I'm interesting to see what's the effect of such high concentration of DTT, also its very difficult to repeat such experiment, in this case I'm looking for whole proteins list in gel and not for specific one so if there is no serious or significant effect I will accept my gel.
Hi there,
1M is so much above recommended DTT concentration... Just have a go and tell us.
OK. In this case, I'd repeat the experiment with correct or even without DTT.
Eric: DTT enable commonly to make reduction of a typical disulfide bond via two sequential thiol-disulfide exchange reactions. However, the reducing power of DTT might be limited to pH values above 7. Thus, you can try to quantify your experimental protein with pH (above 7), and see! GOOD LUCK!
Hi, I also agreed with other replies that 1M is too high concentration for regular case of SDS-PAGE. However, if your target has some special characters, maybe it's a need.
To quantitate protein amount and avoiding DTT interference, you can try to prepare buffer without DTT to solubilize your protein sample first, and perform your protein quantitation assay. After you calculate protein concentration, then you can add DTT solution separately later before loading into the SDS gel. Good luck!!
This is uncommn high concentration of DTT Isuggest if you cannot lower DTT concentration you add the same DTT concentration to control sample and also to standards proteins in your SDS gel and see it effect in these with and wiyhout 1M DTT
good luck
Please try 2-D Quant Kit, GE Health Care, it can tolerate DTT up to 1%.
See the link for details:
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314729545976/litdoc28954714AE_20110830215136.pdf
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