You may check on the hot topic discussion following the question: “Comparison of RT-PCR vs Western blot for tissue protein expression?” asked by Timothy Angelotti from Stanford University. You’ll probably found a few ideas that could help you to clarify the problem of comparing PCR and western blot results.

Hi Muhammad,

there is several basic things that you could do :

-Checking your antibody by adding the positive cell line you mentionned

-adding serum stimulated vs serum starved to see whether this impact your protein expression/translation

-protein could be alternatively translated but quickely degraded thus blocking proteasome might be a way to verify this.

For more general aspect, protein translation could be regulated by p70S6 kinase that is itself regulated by serum, so yes serum could impact protein translation although this is rather cell type dependent. But it your specific situation this should raise a question since mRNA is up-regulated upon starving so what could be the benefit for the cells to up-regulate a mRNA and not allow its translation (if the goal is to have a serum responsive gene??)

Hope this helps a bite

Best regards

Thanx Antonio....Almost same discussion there....But .No final conclusion, however, was worth reading....My protein is transcribed by NF-kB and works as a transcription coregulator. So I think there is no question of halting at mRNA level........

@ Anne Blangy.....This is also a problem....Because the gene is regulated by NF-kB and MG132 or any other proteosomal inhibitor inhibits NF-kB pathway. I have already confirmed the activation and inhibition of NF-kB and the regulation of the gene by employing lactacystiene, LLnL and Bay11-7082....However, I havn't checked the protein expression under these conditions....but giving it a look is of course a good option though...

@ Daniel Bouvard ....The positive control gives very clear result..... I haven't checked p70S6K but I think I should check it.....Also I have done serum reconstitution of the serum starved cells...Upon serum reconstitution the RNA expression is restored to basal level....Furthermore Because the gene is regulated by NF-kB and MG132 or any other proteosomal inhibitor inhibits NF-kB pathway. I have already confirmed the activation and inhibition of NF-kB and the regulation of the gene by employing lactacystiene, LLnL and Bay11-7082....However, I havn't checked the protein expression under these conditions and I think I should give it a trial......Thanx...

Please just keep in mind that MG-132 will also induced a UPR unfolded Protein response that should ultimately lead to protein translation repression. So this experiment should be though in a way that you blocked proteins degradation of translated proteins (all is true for protein sensitive to UPR-induced repression).

Protein translation may be impaired when amino acid content is perturbed - may your serum deprivation affect this? See Trofimova et al., 2012; Araújo WL et al., 2013 and refs therein

@Danial Bouvard ...I conclude I should block proteosome and should check protein expression any way....

@ Victoria Bunik....Thanx for referrring to interesting articles... I believe the media with out serum contains enough amino acids to support cells machinery so I think that will not be a major factor here but still the removal of growth fcators or at least a probable decrease in amino acid in the form of serum removal may however, decrease translation upto some extent....

Hej, yes I think it's worth to get a try

Best

Western blotting may influence the native protein and harm the proper interaction with this particular antibody. Can you do the staining with the antibody directly on slides of frozen cells to allocate specific binding or can you use other gel electrophoresis procedures than western blotting, leaving the native protein intact like isoelectrofocussing or SDS free treatments. Another possibility is using other antibodies for this protein, being active on another part of the protein giving better staining with Western blotting. This may also help you in this respect.

The first question to aswer is does the antibody work for your cells, you say it work for other cells, but that does not mean that is will work for this particular ones. Do you have another antibody against this protein that you could use?

Serum deprivation is no problem because the amino acid are part of the medium (unless you use medium for metabolic labeling). If you have a cDNA for you gene of interest it could be a good idea to check if you can detect the protein after transfection, it would not mean that western blot necessarly work for the native protein but at least it could not be excluded.

I think that the problem is the antibody recognition for your protein

For Western blot, you need to test antobodies using positive control cells. If the selected antibody works well, but the protein expression level of your protein is too low, suggest to use immunoprecipitation method to get enrichment of your specific protein.

@ Geneviève Bart ·....We do have plasmid DNA of the same gene but unfortunately that is HA tagged and the antibody can not detect it.....

@Willem Schoonen....Because we have detected the same protein in another cell line, so I don't think the protein-antibody interaction is hindered under normal SDS-PAGE. This antibody is specific for Western and IP only, so If I have to check immunocfluroroscence then definitely I have to switch to another anitbody. And the final point that you mentioned about epitope differences is something that I need to dig out further, because I am not very much expert in antibody engineering...Any way thank you for your for your sincere suggestions...,.....

@ Orlando Vargas-Sierra .....Thank you, that could be a problem but I want to follow few alternative strategies discussed here, then I will think about changing the antibody...

@ Bojiang Shen....Excellent point....My antibody is also recommended for IP...so immunoprecipitation of protein can increase the amount, and I think that will make the detection a bit easier. If my antibody is OK then I hope it will work or the other way round....

Thank you all for your suggestions...I conclude

(1) Inhibition of proteosome and checking the protein level by Western

(2) Immunprecipitation of protein followed by Western

(3) Changing the sample preparation procedure (Non-denaturing condition) and Western and finally If I am not successful then

(4) Looking for another antibody....

Indeed my manuscript has almost accepted but I am stuck in producing the protein band, as It is required in the revised version of our manuscript....Thank you again...

Muhammad, a polyclonal antibody could be useful in detecting your protein, if you could find one. May need to improve/increase washings after incubations (primary and secondary) to get a good clean labeling for documentation. If the epitope of your monoclonal is lost during blotting, it is very likely that other epitopes remain detectable with a polyclonal antibody.

@ Maria Villacres ....Thanx for suggestion...In fact the antibody that I am using is polyclonal but probably of poor quality...Any way I have done little improvement regarding my experiment/data during last couple of days...However, I just need few more trials to make it clear and confirmed....

Perhaps I am joining the party too late. Was the problem solved?

The rule of thumb is that serum withdrawal suppresses cap-dependent mRNA translation. You may have the same level of mRNA but serum withdrawal will halt translation and this would explain why you see less to none of the protein whereas mRNA is still there.

If your protein is degraded via ubiquitin-proteasome dependent mechanisms (all short-lived and majority of long-lived proteins), then treating cells with proteasome inhibitors (e.g. bortezomib) may stabilize the protein of interest and you will see the band.

Also, If it is HA-tagged, then use anti-HA antibody (depending on number of HA-tag copies you could have an increase from 3 to 9KDA in the size of your protein).

@ Olga Issaenko.........Though it is little late but still your information is worth reading. Yes I have solved my problem, and I detected protein band, but the expression was little lower than I was expecting, (probably b/c of the reason you explained). The protein was endogenous, so I couldn't use HA antibody....Any way thanx for your explanation.