Hi!

The DTT is used as reducing agent for disulfide bonds of proteins while SDS is a detergent, thus SDS is not replacement for DTT. And depending on what you want to do with your proteins afterwards, adding a detergent might be a really bad idea.

Hie! why you want to replace DTT with SDS. SDS can denature your protein..you can try sarkosyl around 0.2% and triton x 100 in 0.1% combination instead of SDS.. This can resolve your problem..try it and give your updates..

all the best..

hi, I agree with Andrei because SDS and DTT are very different molecules. Anyway if you want to prepare a strong lysis buffer you should add a detergent (there are a lot, ionic, cationic, switerionic...you can change the proportion depending on the sample, plant wall, bacterial, human cells, etc), also you need a protease inhibitor and reductor agent to avoid proteases activity, in addition also you need some salts (Tris, PBS, NaCl, etc) to adjust pH and these can also help in the cell lysis.

I recommend you to probe different buffers and conditions, there are a lot of bibliography and commercial books for that.

Good luck and regards

For sonication usual buffer as you planned for purification will be ok. To enhance the cell cracking you might add lysozyme. It works on the peptidoglycan of bacterial cell wall and enhance cell crack. Protease inhibitors can be used to protect you protein of interest from degradation from endogeneous proteases; now a days complete protease inhibitors are available. I use 2 Roche mini complete inhibitors for 20 ml of lysis buffer. Furthermore you can also consider to use Dnase1 if the lysates are too viscous. But as other scientists talked; SDS is really fatal for your protein; because it is a denaturing agent.

A word of caution, SDS will denature your proteins so in case you need your proteins in their native form, then you will have to avoid it completely. Proteins are very sensitive

DTT, beta-ME and TCEP are reducing agents. SDS, Tween-20 etc. are detergents which will denature your protein of choice. They belong to a completely different category.

Knowing the species of bacteria, purpose of your protein extraction and the problem you've got would help.

As others have explained, SDS is a detergent that will denature most mesophilic proteins. Any detergent will have this effect to some extent. Detergent is essential for solubilising membrane proteins after you've isolated the membrane fraction, but for soluble proteins avoid it! If you need reducing conditions, DTT or betaME would be the first choices. Lysozyme may help beak the cell down but is less effective on gram-negatives like E. coli than gram-positives. Maintaining some osmolality, with salts and sucrose or glycerol, can help stabilise some proteins. Divalent metal ions (eg Ca, Mg) are needed by some proteins but can enhance protease activity - typically, either a metal ion or EDTA gives the best results, depending on your target protein. PMSF as a protease inhibitor is almost always worth including (unless you're after a protease). If you're not getting enough protein out, increase the sonication power &/or time or try another breaking method (French Press, freeze/thaw with osmolar shock etc)

DTT and SDS are used for different purposes in protein buffers so you could not replace them reciprocally. Standard methods to obtain native proteins imply the utilization of buffers at pH 7 or 7.5 essentially containing Tris-HCl and salt (like NaCl). If you are interested in obtaining a particular protein and have problem in getting it in your lysate you can try to change pH or salt concentration to improve solubility in the buffer. In any case, I would like to suggest you to add protease inhibitors (some people use DTT or other reductants at very very low concentrations to prevent protease activity in native lysis buffer but it would be best to work at low temperature and use other inhibitors like PMSF).

All the best,

GG