I need to understand if an antibody I'm using is specific or not.
The antibody is not validated.
In immunoblotting, the antibody detects a prominent band at the right molecular weight and two further nonspecific bands.
In negative control protein lysate, the band at the right molecular weight disappears, while the two nonspeific bands are still detected.
In immunofluorescence, I have an intense staining in the right cell localization.
Unfortunately, the blocking peptide for this antibody is not available.
Could the full lenght recombinant protein targeted by the antibody be suitable as blocking peptide?
PS: no data from litterature are available about the expression of the protein of interest in my cellular system.
Thank you.
Brandie M. Cross
thanj you for the answer.1) No, I tried only in western blot. Unfortunately I don't have in my lab living cells not expressing my protein .
2) This is my question: can I use my whole protein as the "peptide block"?
Dear Nicola
i think that there is a difference beetween specificity and neutralizing.
specificity is the ability of your antibody to recognize only your protein and not others and from the an results it seems not very specific because it recognize to bands also in the negative control lisate. What are negative control? empty cell lines? are you sure that this cell lines intrinsically do not express your protein or a similar variant?
neutralizzation is the ability of your antibody to block the function of the protein but may happen that one antibody is specific but not able to do it because bind on a epitope far from the catalitic or interaction site of your protein. This not means that the antibody is not good, depends what you want do with it.
to check if your antibody is netraulizing you need to set up a functional assay for your recombinant protein and check in addiction or pre-incubation with the antibody may inhibit or not this function but the feaaibility of it depends a lot from your protein function.
ciao
Manuele
Dear Manuele,
thank you for the answer.
I meant specificity.
I'd like to know if the band at the right molecular weight is specific (at that mw the antibody doesn't detect bands in negative control cell lysate)
Nicola
The non-specific bands detected in WB, often minor, are usually artifacts related to the use of cell lysates (or impure proteins) containing cytochrome C or IgG receptors ... The differential between positive cells and cells negatives makes it possible to guarantee the specificity of marking. In publications one is careful not to show these artifacts in order to avoid having to give explanations. As for the blocking of the labeling by the specific antibody, it is imperative to put very large quantities of target proteins to obtain a specific inhibition: of the order of 10 to 100 times the amount of antibody applied. On the other hand, the antibody having a high affinity for the whole protein as for the synthetic peptide, it is preferable to use the entire protein when it is possessed.
- Badges
- Science method