I am interested in assessing the expression of some growth factor (e.g. TGF-beta 1) or other marker (e.g. PCNA, CD31). I want to know the difference in term of significance for assessing the same marker by western blotting and immunohistochemistry (IHC). Is there significant difference for assessing the expression of same marker by these two different techniques?
Hi Vinay,
Yes, there are.
Primarily these two methods detect target proteins depending on the antibodies you are using - at this point they are quite the same thing. However, IHC refers to the immunolocalization of a given protein in a slice from a piece of tissue. By using western blotting you are able to separate proteins by molecular weight and further semi-quantify them in a PVDF or nitrocellulose membrane by using the antibody against the protein of interest.
Both methods are time consuming but I would say that western blotting is way far from being nicely reproducible even for very skilled personnel.
I guess it really depends on your question, if you want to show the expression and localization of a protein in a specific tissue, IHC is definitely a great option. With IHC you can also quantify the expression of you target by number of fields analyzed, but always keeping in mind the limitation of this kind of quantification; if you want to see overall expression in a tissue lyzate then western blotting would do the trick, though you have to keep in mind that you would be semi-quantifying the target protein and you will always have to run a housekeeping protein (i.e. actin or GAPDH) to correct your values relative to.
If you want to have a more accurate measure of the protein of interest I would recommend ELISA instead.
There are several good guide books at suppliers website, for instance, ABCAM have easy to follow general guidelines for several techniques.
The rule of thumb, I would say, is choosing a good antibody and knowing the limitations of these methodologies.
Hope you find the best option for your experiments.
Regards
Hi Vinay,
Yes, there are.
Primarily these two methods detect target proteins depending on the antibodies you are using - at this point they are quite the same thing. However, IHC refers to the immunolocalization of a given protein in a slice from a piece of tissue. By using western blotting you are able to separate proteins by molecular weight and further semi-quantify them in a PVDF or nitrocellulose membrane by using the antibody against the protein of interest.
Both methods are time consuming but I would say that western blotting is way far from being nicely reproducible even for very skilled personnel.
I guess it really depends on your question, if you want to show the expression and localization of a protein in a specific tissue, IHC is definitely a great option. With IHC you can also quantify the expression of you target by number of fields analyzed, but always keeping in mind the limitation of this kind of quantification; if you want to see overall expression in a tissue lyzate then western blotting would do the trick, though you have to keep in mind that you would be semi-quantifying the target protein and you will always have to run a housekeeping protein (i.e. actin or GAPDH) to correct your values relative to.
If you want to have a more accurate measure of the protein of interest I would recommend ELISA instead.
There are several good guide books at suppliers website, for instance, ABCAM have easy to follow general guidelines for several techniques.
The rule of thumb, I would say, is choosing a good antibody and knowing the limitations of these methodologies.
Hope you find the best option for your experiments.
Regards
Hello!
I agree with everything that Felipe said. The principle of detection is more or less the same. With Western Blot you separate proteins by size, and semiquantification is possible. The great advantage of IHC is that you see where in the cell your protein is localized. This is of special interest if looking at proteins that change localization for example upon activation (eg. transfer from cytoplasm to nucleus, or from ER to cell membrane). If you just want to show wheter a protein is present or not, Western Blot might be the method of choice.
What we did previously, is to use Western Blot for antibody testing regarding specificity and sensitivity. That means, if an antibody resulted in one clear specific band on the blot at the expected size and not showing any unspecific bands at other sizes, the antibody was deemed useful for IHC. On the other hand, if we got an unexpected band pattern, the antibody was not further used for IHC, because in the end you would not know what exactly you have stained.
So again, chosing a good and specific antibody is crucial for IHC and western blotting might help to find one.
Good luck!
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