If the result is idean and no human or technical error, would coomassie and immu
ID: 205840 • Letter: I
Question
If the result is idean and no human or technical error, would coomassie and immunoblot show same result with same band size and intensity? What would be the difference between these two in result? Does immunoblot show all the same size of band at spacific kDa? If the result is idean and no human or technical error, would coomassie and immunoblot show same result with same band size and intensity? What would be the difference between these two in result? Does immunoblot show all the same size of band at spacific kDa? What would be the difference between these two in result? Does immunoblot show all the same size of band at spacific kDa?Explanation / Answer
1. Coomassie blue staining is a measure of protein concentration. This dye binds to all proteins regardless of size. Hence, a band in Coomassie blue staining will be made of all proteins that have the same size. On the other hand, immunoblots will show specific band that correspond to the molecular weight of the protein. This is because of specific interaction between antigenic epitopes of protein and antibody. Hence, the band on immunoblot will be thinner and more specific as compared to the Coomassie blue band. The intensity will depend on the staining procedure. If signal enhancement is preformed, then the band will be more intense on detection. However, if no signal enhancement is performed, then the band will be of low intensity.
2) Coomassie blue shows more diffuse and generally larger bands than immunoblots. If the protein is expressed in low amounts, Coomassie may show a large band but immunoblot may not even be able to detect it or detects a faint low intensity band. Conversely, if protein is in high concentration, band sizes may be equal in both types of detection.
3) The size of the protein does depend on the molecular weight of the protein. However, due to posttranslational modifications, proteins may migrate either slower or faster in a gel. Protein degradation may reduce the molecular size of the protein. Glycosylation cause the protein to exhibit a higher molecular weight than expected. Such proteins will run slower on the gel. Protein aggregation, residual disulphide binding or incomplete denaturation will also cause the band to have much higher molecular weight than expected. If there are splice variants present, antibody will stain them. However, these splice variants will have smaller molecular weight than expected.
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