(best cell lysis method for) protein expression analysis by western blot - (Mar/01/2013 )
Hello everybody,
After transfecting 293T cells by several construct vectors expressing a certain type of protein, I was able to detect my proteins by Immunohistochemistry (IHC), but not by western blot, although using the same primary antobodies fro both western and IHC, and getting +ve result for the +ve control as well.
Do you think that the method of lysing the cells to extract the proteins could affect my ability to detect them by western blot? I'm using a method dofferent from that used for tissues.
or is it the quantity of proteins expressed? so that Immunohistochemistry is more sensitive to small protein amounts.
or the post-transfection time is a factor of the expression of the proteins?
of course it's not a matter of protein stablity because otherwise I couldn't have detected it by Immunohistochemistry.
Nor it's a problem of primary antibody concentration because I increased it and still don't see any specific bands on western blot.
thanks for helping me
molecularbio on Fri Mar 1 10:26:38 2013 said:
Hello everybody,
After transfecting 293T cells by several construct vectors expressing a certain type of protein, I was able to detect my proteins by Immunohistochemistry (IHC), but not by western blot, although using the same primary antobodies fro both western and IHC, and getting +ve result for the +ve control as well.
Do you think that the method of lysing the cells to extract the proteins could affect my ability to detect them by western blot? I'm using a method dofferent from that used for tissues.
or is it the quantity of proteins expressed? so that Immunohistochemistry is more sensitive to small protein amounts.
or the post-transfection time is a factor of the expression of the proteins?
of course it's not a matter of protein stablity because otherwise I couldn't have detected it by Immunohistochemistry.
Nor it's a problem of primary antibody concentration because I increased it and still don't see any specific bands on western blot.
thanks for helping me
Not all antibodies work well for both immuno-staining and western blot. What did the company specification sheet say about applications of that particular antibody?
By +ve controls, what do you mean? Cell lines that already express your proteins of interest abundantly and you used the same protocol in both staining and western blot - and got positive outcomes?
Immuno-staining is definitely more sensitive than western blot (unless if you've mistaken background/non-specific staining as a positive one in immuno). Do you know if your plasmid construct contains a GFP tag or His tag - which will determine your transfection efficiency. Have you selected your clones using antibiotics against resistant markers? It's important to note the efficiency of your transfection as this will determine your protein expression.
hello dear,
thanks for ur feedback
those antibodies actually I already used in western blot before and work very well: however they worked on proteins from tissue extracts not from transfected cells' extracts.
+ve controls are cells transfected with a vector expressing another protein but having the same tag as my proteins (myc), so the antibodies of the tag work.
my plasmid constructs have myc tag, and the point is that I can detect myc more than the protein itself (for which I have antibodies but without a +ve control), in immunohistochem,
Moreover, transfection is working because I have a GFP construct as a control of transfection.
yes, my clones ofcourse are selected and subcloning itself is a successful step, the problem is either in the expression of the protein, or its stablity or method of extraction for western blot,
Do you know a protocol for membrane proteins' extraction? they could be more sensitive to denaturing conditions than cytosolic proteins maybe..