Proteins troubleshooting

A silver stain is used when proteins exist in a very small quantity or when analysis of as many bands as possible created by separation techniques is desired. One positive apphcation of silver stain is its sensitivity. A drawback of the silver stain, however, is that it is more complex and often requires more troubleshooting to obtain the desired results. 

Table B1.1.1 Troubleshooting Guide for the Modified Lowry Protein Assay... 

Table B1.1.3 Troubleshooting Guide for Biuret Protein Assay...

Table B1.1.4 Troubleshooting Guide for Coomassie Plus Protein... 

The main disadvantage of all Bradford-type protein assay reagents is that they are not compatible with surfactants at concentrations routinely used to solubilize membrane proteins. With some exceptions, the presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent. Table B1.1.4 is a brief troubleshooting guide for this technique. 

Longer equilibrations in transfer buffer prior to electrotransfer may extract too much SDS from the gel and hence reduce transfer efficiency. This equilibration step will remove excess SDS and prevent the gel from swelling during transfer. The negatively charged SDS allows proteins to move toward the transfer membrane in an electric field. On the other hand, too much SDS decreases binding of protein to PVDF membranes. Therefore, the amount of SDS is critical for transfer yield (see Critical Parameters and Troubleshooting). 

Bjerrum, O.J., Larsen, K.P.. and Heegaard, N.H.H. 1988. Nonspecific binding and artifacts—specificity problems and troubleshooting with an atlas of immunoblotting artifacts. In CRC Handbook of Immunoblotting of Proteins, Vol. I Technical Descriptions (O.J. Bjerrum and N.H.H. Heegaard, eds.) pp. 227-254. CRC Press, Boca Raton, Fla. 

Most of the mentioned troubleshooting tools will work with other silica-based columns. With normal-phase columns, you obviously need not worry about bonded-phase removal, but silica still dissolved at high pH and high salt concentrations. Polar materials like some proteins adhere very tightly and require... 

It is highly recommended (especially the first few times this method is used) that fractions be saved from each step of the purification procedure and small aliquots of these fractions analyzed by denaturing PAGE. Problems with the quality of the HMM protein, the transcription yields, binding to the column, etc. can be detected and appropriate troubleshooting conducted. 

Jones, K. D. (1999) Troubleshooting protein binding in nitrocellulose membranes Part I Principles. IVD Technol. 32... 

Separations of proteins or nucleic adds are commonly carried out using gradients of increasing ionic strength. It is a good practice to monitor these gradients with a conductivity meter. This provides added information for troubleshooting. 

---