I tried two antibodies and one show stain in the cytoplasm and the other one in the nucleus. Why?
(Blue - dapi; red - mitochondria; green - protein of interest)
And why is hard to find a good antibody for some proteins?
If both antibodies you tried were specific and you observed them in two different locations as you mentioned, you should for sure question the specificity of one or both the antibodies you used.
Also, where did you expect to see the staining at the first place?
Vahid M. Harandi i actually hoped to see it overlap mitochondria.
I added some images of the staining: for the one that stains the nucleus I got one unspecific band and the one that stains the cytoplasm i didnt get any unspecific band.
hi
You have taken beautiful photos.
Please name the antigens and antibodies. Another point is whether your antibody is monoclonal or polyclonal?
good luck
Reza Hadavi Thank you! Blue - dapi; red - mitochondria; green - protein of interest. Both antibodies are polyclonal
Ivana Zheng Maybe I didn't fully understand what you meant but did you do Western blot as well when you are referring to the bands?
Were your antibodies raised against synthetic peptides (i.e. despite being polyclonal, they are recognizing only a small portion of the whole protein) or against the whole protein?
If there were no errors or accidents (interchanged ABs, etc.), there might be posttranslational modifications (e.g. phosphorylations) which are different in different locations and result in binding / not binding of one of both antibodies.
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