We did sds page using artemia protein looking for hsp-70. We were instructed to
ID: 6719 • Letter: W
Question
We did sds page using artemia protein looking for hsp-70. We were instructed to cut the protein bands out of the gel and use electroelution to separate the proteins further(?) In the elution process we used membrane caps that MW weight cutoff was 3500 Da. Next we ran electrophoresis gel again and then performed a western blot to identify protein. I guess I am stuck on why electroelution and what exactly is the differences. I understand the sds page but am lost on the electoelution and its protocol. Like why we use the reagants we do and what do we want to see? I am lostExplanation / Answer
In SDS PAGE the following reagents are generally used which show properties like following-
Tris – used as buffer.
Glycine – to maintain the pH.
Acrylamide – polymerization substance.
Bis acrylamide – cross linking agent.
Sodium dodecyl sulphate – to denature native proteins to polypeptides.
Ammonium per sulphate – initiator for gel formation.
TEMED – polymerization substance.
For staining purpose reagents like bromophenol blue, glycerol, and coomassie blue are used which are dissolved in solvents like Butanol and Dithiotheritol.
After the elctrophoretic run the gel is cut for protein bands which are identified by visualizing under UV.
These protein bands will be subjected to electroelution. In this the minced gel is placed in dialysis bag. The salt and SDS are removed through dialysis and the protein is precipitated to remove the remaining detergent and salt. After this the protein which is devoid of impurities is used for analysis.
In this way the protein study can done by using both the SDS PAGE and electroelution.
In SDS PAGE only the protein of interest is isolated from mixture of protein molecules. But in electroelution the isolated protein is purified.
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