klenow

I think the question is if with these antibodies you detect that the endogenous protein has a lower molecular weight band. If this is not the case and you never detect endogenous levels, it is not surprising to have differences between the stimate MW and the real one. To be sure, you can check the overexpression construct to make sure the sequence is correct.
If everything is OK and the endogenous protein does have a lower MW could be that, as bob1 suggested, overexpressed protein is having some posttranscriptional modification... however, I would try to do RNAi against your protein of interest, because maybe the antibodies are not sensitive and/or specific enough. I had a similar problem, but the difference between real and expected MW was too much to have doubts. The antibody only recognised overexpressed protein (single band) but when non-transfected cell extracts were used, the antibody recognised a non-specific band... Just to be sure I performed RNAi against that protein and confirmed the "endogenous" protein recognised by the antibody was not the good one...

Thanks a lot Andreea,
C-terminus is my only choice because a tag in the N-terminus influences localisation and function of the protein... it is a secrerted protein and there is a signal peptide, so N-terminus tag is not an option. I am making, just in case, a new construct having an internal tag between the signal peptide and the N-terminus of the secreted, processed protein. In the meantime, I will try with UREA and I hope it works.
Best

Another test (but requires some work and maybe is not necessary) is to build a miRNA spongue to your candidate miRNA and check what happens with your candidate gene when the spongue is transfected into cells.

The plasmid should contain some gen that confers resitance agains an antibiotics (puromycin resistant). If this is not the case, you should clone your cDNA into a more appropiate vector because although you can do stable cell line with your plasmid, you will have to check for expression everytime you want to use the cells (and without a visual marker that means to perform PCR or better Q-PCR if you want to compare levels and/or Western blot)... In my opinion, instead of doing all that stuff before any experiment, I would prefer spend one week and clone the cDNA in another vector...
Best,

Hi,
As bob1 says, miniprep refers to a purification of plasmid DNA... not inserts or digested plasmids. After electrophoresis I use to check the correct size of both insert and digested vector (use a low-power UV lamp...) and then cut the correct bands. After that, I use a kit specifically designed to purify double strand DNA (from agarose or from PCR reactions... ). I usually check the DNA concentration and then I perform the ligation. For that I usually try 1:3 to 1:10 molar ratios (digested plasmid:insert). You can find ligation and transformation protocols in the forum. Then, you have to see if the ligation worked and select some colonies. I like to confirm that the colonies have the proper insert in the proper vector by doing colony-PCR (basically, you amplify a region containing your insert and some part of the vector). From the positive clones, I culture some of them, do a miniprep, keep a glycerol stock and sequence them.