Dear Colleagues,
I am trying different protocols in order to find the best one for protein extraction in my tissue. Some extraction solutions contain SDS, DTT and CHAPS. is it mandatory to remove the detergents if I am simply going to perform classical western blot ? And can I stock my extracts in the -80°C by the time I can get a detergent removal solution or column? And if I need to reduce these detergents what is the best product to use is the Pierce detergent removal spin columns the best thing to do?
Thank you in advance
What is the nature of the extracted protein? It's not necessary to remove contaminating detergents including SDS. However, for some extracted enzymes SDS may inhibitors their activities. Eg.lipase is inhibited by SDS. The dialysis is an easy option or pass the extract through PD-10 or Sephadex G-10 column and collect starting fractions containing protein. SDS and other detergents shall stay in the column.
Fore removing detergents you can use dialysis or amicon with proper cut of.
If your going to store your pro it is better to use glycerol.
But about western blot it depends to your purpose.
You want just check present of pro or wanna check the structure too?
For typical Western blotting, there is no need to remove detergents or reducing agents if you are just looking at protein presence and size. For this type of Western, you typically want to use SDS and DTT or 2-mercaptoethanol in your sample buffer. You also use SDS in your running buffer (referred to as running denaturing gels under reducing conditions).
As Shamsher mentioned, if you have some application involving enzymatic activity or protein-protein interactions, you would need to remove the detergents and reducing agents to try and recover the activity or interactions (referred to as running native gels). Removing these is also important if you are performing HPLC or mass spec analysis. The reagents mentioned can be removed by dialysis or gel filtration, however, take care if you have Tween, TRITONX, or NP-40 as they are not easily removed by these methods.
Removal of mentioned substances are always depends on purity and activity of protein of interest (early or later). Sometime getting rid of SDS is difficult just by dialysis. Better is PD-10 or Sephadex G-10. Short storage in -80 or -20 is ok but that too depends on what you want to do with your protein in future. I mean for what purpose you want to use. No need to remove mentioned substances for western blotting. But if you want to do Native PAGE and then western blotting so in that case you have to get rid of these chemicals. I hope you got answers, all the best.
Ok. Then no problem, you can carry on SDS-PAGE and western blotting without removing mentioned chemicals. You may see some kind of artifact, that will be of detergent. Don't worry you will understand the difference between band and artifact (Color, sharpness and intensity difference).
If your work flow just ends in western blotting you do need worry about detergent remnants whatsoever.
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