Sample denaturation is one of the steps that should be carried to prepare the proteins for SDS-PAGE. Should we carry this step out for all proteins? Or can some proteins be affected by this step?
Other question, I am using a reducing buffer to prepare the loading buffer, but these days we don't have the reducing buffer, so I plan to use DTT (dithiothreitol) 1M what is the ratio of DDT: loading buffer I should use?
The loading buffer is not a denaturing agent. DTT is "only" a reducing agent. SDS is a detergent and mainly stabilize the denaturation. To denaturate the proteins, you need the heat your sample or the use urea at high concentration, at room temperature.
No, you do not need to denaturate your sample to perform a migration in your SDS-PAGE gel. But you need SDS to negatively charging the proteins of your samples.
Without denaturation (as example heat), you might have adverse effect such as modification of apparent protein size due to secondary/tertiary structure even in presence of DTT. In addition, this size modification might be partial, and you might observe a smear of your protein of interest, but not always...
DTT is a strong denaturant capable of reducing tertiary and quaternary protein structures by cleaving disulfide bonds. SDS does not reduce disulfide bonds, but aids in the reduction of the tertiary and quaternary structures by coating the protein with a negative charge (prevents refolding). It also reduces non-covalent bonds. Many loading buffers contain beta-mercaptoethanol as it also cleaves disulfide bonds and aids in the aqueous solubilization of proteins. It may be necessary to add this to your loading buffer in addition to DTT.
Broadly, the sample denaturation step should be specific to the protein of interest. Hydrophobic transmembrane proteins can require specialized denaturation. Many enzymes should not be denatured/reduced. If you're looking at the aggregation state of a protein, denaturation and reduction would be detrimental. I'm sure there are many other instances. Perhaps you need to look at Native PAGE?
Hope this helps.
I've had some problems with membrane proteins. Sometime they become agregated after boiling and migration of the protein in SDS-PAGE is not according to their molecular weight. To avoid this problem I use to incubate the samples with Laemli-SDS buffet (with DTT) at 37ºC for 30min. So, the answer is yes, there are proteins that are affected by this step
The loading buffer is not a denaturing agent. DTT is "only" a reducing agent. SDS is a detergent and mainly stabilize the denaturation. To denaturate the proteins, you need the heat your sample or the use urea at high concentration, at room temperature.
No, you do not need to denaturate your sample to perform a migration in your SDS-PAGE gel. But you need SDS to negatively charging the proteins of your samples.
Without denaturation (as example heat), you might have adverse effect such as modification of apparent protein size due to secondary/tertiary structure even in presence of DTT. In addition, this size modification might be partial, and you might observe a smear of your protein of interest, but not always...
I usually add PMSF and DTT in my lysis buffer instead of sds but before protein loading samples should be heated at least for 10 min in the presence of SDS
bur if you need your protein in native form e.g for enzyme assays and so never use SDS nor in the buffer or gel
PMSF is a protease inhibitor not a detergent. This product has no effect in the cell lysis
yes I know ... I add PMSF as protease inhibitor in the presence of DTT and this gave me the best result ,,, I add only SDS in the loading buffer and heat for 10 min right before gel loading ... thanks
100 mM DTT in the final sample is generally adequate for most proteins. It is more powerful than BME since it can reduce 2 -S-S- bonds per DTT molecule as against only one by BME.
Thanks for valuable and helpful suggestions and comments,
Dear Anant Dave, Our labmate there are using DTT 200mM, is this one ok?
I mean by denaturation step (using heat), I know DTT is a reducing agent, but if I am targeting cellular protein (signling pathway's protein), should I use this step before I run SDS-PAGE?
I am using heat at 95 C for 5 min. for SDS-PAGE. Primer structure of proteins should be appears that SDS cover around of proteins. if you want to do you can use another denaturing agents such as urea
HI Bashir, DTT is a reducing agent and is generally added in excess. The actual concentration of DTT would also depend on your sample concentration in the sample. It is hard to say what concentration is enough, unless you try it.
Again the choice of this method depends on your final application and information required. As pointed out by others here the components of the PAGE loading buffer includes a denaturant usually SDS. The denaturant would irreversibly denature your protein and hence if you are interested in an bioactivity from this protein, this method is not suitable. As Recep pointed out above you could substitute SDS with Urea too.
The other component in the loading buffer affecting the protein structure is the DTT or BME. These reducing agents help in the unfolding of the tertiary structure by breaking disulfide bonds.
So if you know that your protein has any disulfide bonds and if you are interested in simple qualitative comparison, you may wish to follow this step. However if you do not know whether your protein is likely to contain disulfide bonds, better to run both reducing and non reducing gels.
In the non reducing gels, do not heat the samples at any stage. Just suspend the sample in buffer without the reducing agent and use for analysis.
Hope this is helpful,
Regards,
Anant Dave
- Badges
- Science method