When the primary-secondary antibodies are used to identify proteins using HRP or alexafluor, how to make a choice whether i should extract the proteins, perform electrophoresis and blotting and then use primary followed by HRP tagged Secondary antibody, or i should simply tag fixed cells with primary antibody followed by secondary antibody tagged with alexafluor. Its a very basic question, but kindly help me understand.
Hi Priish,
If I am getting your trouble correctly, you meant by why to bother doing laborious western when IHC is available? right!!.
So it depends on my things. (1) If you want to know the spatial localization of your protein in a specific cell or tissue (such as protein X is expressed more in cell membrane rather than nucleus) you will prefer IHC over western. (2) As Namita said, western is way more quantitative, while IHC can only give you the answer in " more and less" (qualitative). So let's say if you want to measure what is the increase in protein X concentration upon some treatment, you would choose western (in terms of percentage). (3) Some antibodies can only detect the denatured proteins, so if you are stuck with those antibodies- then you have to choose western. (4) Sensitivity: western is way more sensitive than IHC. So If you are not seeing any IHC staining for a particular protein, sometime you have to confirm by western that whether indeed protein is not expressed there or it is expressed in low quantity that you IHC was not able to detect.
I hope this would help.
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