I have cloned my gene of interest in pcDNA3-CMV-APEX-NLS and this construct was used to transfect HEK293T cells.pcDNA3-CMV-APEX-NLS was used as a control during transfection.I have performed biotinylation expt.(reference-RHee et al,2013) after 24 hrs of transfection. I have tried to visualize the protein using silver staining after streptavidin pulldown.I could see a very faint band of my protein along with the background.I have increased the cell density to 16 million cells(at the time of processing) to get a prominent band during silver staining.But it is not working. I would appreciate if you have any suggestions on this. Thanks.
This method (using APEX) biotinylates proteins in the proximity of (i.e. near to) your tagged protein of interest, so that you can identify proteins that are close in space (but not necessarily binding). After pulldown you should expect to have lots of proteins present - this might be the 'background' that you see, which could actually be biologically relevant (see Fig1D of the paper you refer to). To confirm this, if you do the procedure in parallel but without the biotinylation reaction, you should not see any proteins pulled out. If you are concerned that the reaction is not specific enough, then perhaps you need to titrate down the substrate concentration or incubation time, etc.
Depending on the aim of your experiment, there may be more appropriate methods to use. If you are trying to identify direct binding partners, then your protein of interest should be tagged with something else such as 3xFLAG, myc or GFP before performing IP for the tag. If you are trying to purify your protein of interest, then the APEX method is definitely not designed for this kind of experiment.
Thanks Alex.
The problem is specificity.I could not find any specific band in presence of my protein.Lets see if I can do something to enhance the specificity.
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