I had cloned my gene of interest (GOI) into pCMV10-3X-Flag vector for expressing into mammalian cells. Even though the gene has been cloned and sequence confirmed, I am having a problem with expressing it in HEK293T cells. Though immunofluorescence shows ~ 60% efficiency at 8micrograms of the construct, when it comes to checking of protein expression by western blotting I cannot see the band at all by using anti-Flag or antibody against my GOI.
Can anyone suggest the probable reason for it and a solution?
What is the molecular weight of your protein? Very large (>200kDa) or very small proteins (
Hi Daniel.My protein is a nuclear one,size of 21kDa. The transfer time onto PVDF membrane has been standardized with the bacterially purified recombinant protein,which is His tagged;so it being running out during transfer or the chances of not seeing on blots can be done away with.In fact the bacterially expressed one is visible on the blot to a good extent,only when I am trying to do the same from the cell lysate the problem of non-visibility,on the blots,come in.
What do u think might be the reason?
Nice control! That simplifies matters. How do you prepare the cell extract that is loaded on to the SDS-PAGE gel? Is it possible that your GOI isn't solubilized? Some protocols include a centrifugation step to pellet debris after lysis. Nuclei are a major component of this insoluble material.
Are you using the same anti-FLAG antibody for IF and Western?
Ah!!! I had not checked the pellet fraction after lysis. Thanks for bringing it to my notice.I had initially used the RIPA buffer for lysis,and then switched to follow the lysis bufer composition as under,both ways no expression in lysate:
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/a2220bul.Par.0001.File.tmp/a2220bul.pdf
Though I think the buffers mentioned as above will lyse the nuclei as well,but still I will check the pellet fraction.
Try using SDS-PAGE sample buffer as lysis buffer, sonicate at 5-10 microns for 10 sec to break down DNA, load onto a SDS-PAGE gel and perform western blotting. Do you detect your band?
I use a similar protocol as Estanis. If solubility is the problem this should help. Another point to bear in mind is concentration of your lysate. Eventhough Western can be quite sensitive, you will have detection problems if your samples is too dilute. How many cells do you lyse and in what volume of lysis buffer? I would suggest using around 5x10^6 cells per mL.
and provides an unfractionated sample to analyze. If it's a nuclear protein, and it's associated to chromatin, after RIPA extraction the protein will surely be in the pellet
The protein is transcription factor,it is soluble and I have loaded lysate of the concentration of 50 and 100 micro gram,as estimated by Bradford's method;but could not find any expression in either case.
Some thoughts:
1) do you see any band on your gel in the positive control? Maybe the apparent migration is different from the expected size?
2) is it possible that your nuclei are not efficiently lyzed in these conditions? Can you detect histones or another endogenous TF on your blot?
3) maybe your estimates of protein concentration are somehow off? Make sure you check your membrane by a reversible protein stain such as Ponceau S. If you are really loading 50-100 micrograms of protein, you should see a ladder of strongly staining bands
4) sometimes immunofluoresence can give false positives, and the true result is that your plasmid and/or transfection is not working. Do you have a negative control to show that you do not detect signal in non-transfected cells? Also, perhaps you can run another flag-tagged protein (cytosolic, abundant) as a positive control for your Western reagents.
I will keep in mind these things,and repeat the expt once again and let u know about the results.Thanks for the kind help.
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