Hi,I did my qPCR experiment in kidney tissue for alat2 gene ( mitochondrial alanine aminotransferase) and I got 3-4 fold increase as compared to control, but when I am doing my Western Blotting ,I am not getting any protein bands at al.Please suggest any reason or solution for this.,thank you
Often, the main problem is to get a specific and sensitive antibody. Do you have a chance to check your antibody, e.g. by forced expression of your cDNA or has this already been done? A positive control may also help to optimize the conditions (bloking agent, dilution ...).
One problem could be the low level of ALAT2. A PCR signal does not mean that there is much protein. Have you tried to enrich for mitochondria? Are you already using a sensitive detection system?
An increment in the transcription level does not necesarily mean a higher expression yield. It depends on the protein turnover, stability, others.
Of course as Uwe suggest you, the first thing to be sure about is if the antibody you are using is good enough, just incase. Or if this has to be use in native or denatured conditions for a proper detection.
Try to optimise the conditions, especially the concentration of the antibody you are using.
Good Morning,Sir I have separated mitochondria following this paper.(A simple protocol for the subcellular fractionation
of skeletal muscle cells and tissue)Regarding antibody, I am getting good band in my other tissues like liver,brain and muscle but not in kidney.So,what I want to know is that,is it necessary if gene expression is there , then protein expression has to be there in a particular tissue?
Good Morning Suman,
It is always a problem to state that a protein is not present in a certain tissue. But since you have a good antibody you can show that there is either no protein or the protein is below detection. The PCR is very sensitive, you may detect a few transcripts and they be even not from typical kidney cells, but e.g. from blood vessels.
So the reason that you detect RNA but no protein may be due to the low expression. However there is no strict correlation of RNA:protein level sinc there is lot posttranscriptional regulation.
In your case, the paper by Rong-Ze Yang: "Alanine Aminotransferase Isoenzymes: Molecular Cloning and Quantitative Analysis of Tissue Expression in Rats and Serum Elevation in Liver Toxicity" seems to be in agreement with your results.
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