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WB band size and protein prediction is not consistent? Check these 5 points first!


There are tens of millions of laboratory metaphysics, and half of them do not agree with the results.



WB experiment is an experiment that students often patronize. But it is such a primary and commonly used experiment, but there will be all kinds of strange problems. For example, when we identify proteins by WB, the band size we get often deviates from the predicted value. This is suffering from "obsessive-compulsive disorder" students can not bear. If the experiment is wrong or the prediction is not accurate, Xiaobian can't jump out of the screen to save you. However, the following five criteria can be treasured by students. When encountering the deviation between protein band size and predicted value, they can take them out and analyze them.



1、 Endogenous protein or recombinant protein



The problem should be checked from the source, so you should first take a look at whether the sample is endogenous protein or recombinant protein?



If the endogenous protein is not tight, if it is a recombinant protein, then we need to see whether the recombinant protein is the full-length sequence. If the sample is a recombinant protein, but not a full-length sequence, then the band size must be inconsistent with the predicted size.



2、 Multiple isoforms



If it is confirmed that the sample is an endogenous protein, it is necessary to see whether our target protein has other isoforms. We usually check NCBI or Swissprot to confirm this.



3、 Attention should be paid to shear activation



If it is mentioned in Swissprot or authoritative literature that the protein in the sample needs shear activation, then the result will be different from the predicted value.



4、 Polypolymer



If there is a significant multiple relationship between the protein size and the predicted value, consider whether the protein is a dimer or a polymer.



5、 Protein modification needs to be bigger



If the above four reasons are excluded, the predicted value is inconsistent with the experimental results, that is, the protein we are facing is a fickle object, which not only cleaves and activates, but also modifies after translation and expression. For example: such as methylation, acetylation, phosphorylation, glycosylation, alkylation, biotinylation, glutamic acid, glycine, sulfation, isoprene, thiooctylation, phosphorylation of Pan acyl mercaptoethylamination. Proteins in different states will have different modifications. Adding functional groups will definitely make the protein larger than predicted. In daily experiments, glycosylation is a more common modification, which generally leads to a larger band than predicted.



Summary:



1. Determine whether the protein is endogenous or exogenous, and if it is exogenous, whether it is full-length sequence;



2. Swissprot database was used to query whether the protein had other isoforms;



3. Swissprot and literature were used to find out whether there was shear activation, which resulted in the decrease of protein size;



4. Determine whether the protein is dimer or multimer.



5. NCBI and Swissprot were used to check whether there was any modification after protein expression. If there was modification, the protein would increase;



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