Dear Colleagues
I have a colleague who is not expecting an antibody in her lysate to give expression of a protein at about 23kD (owing to gene methylation)
However, the antibody does give bands of a much higher Mr. What is more, there is no positive control on this blot which (would validate the antibody)
Am I right in thinking that unless you have a positive control on the blot which validates the antibody i.e. produces a band of 23KD then you cannot infer that other lysates do not express (by absence of that band). By extension, there is little point in stripping this blot and re probing with actin (?)
Rather a sensible course of action would be to repeat the blot and include a positive control; and then strip and re probe with actin (as a loading control)
Hey Laurence,
sounds very good to me. You need a positiv control just to show that, if there is something, which can be detected specifically, then your antibody binds to it.
You may not have to strip the blot just for actin staining. Actin is so strongly expressed that the actin bands will just overexpose all other bands.
I agree with yours and Marc's assessment in that a positive control is crucial here. I have a couple of questions/recommendations.
1. has this antibody been published before in the same type of lysate you are using by other groups or are you searching an unknown in a lysate for the first time? That would help to determine if this antibody is good or unspecific. If other groups have found this protein in the same lysate you are using than you have a problem.
2. Is the antibody from a reputable company that provides references or citations used by other groups? If you purchased the antibody from cell signaling for example, they sell positive controls to use in your blots if you speak with a specialist.
3. Does your antibody come with a pre-absorption peptide control? if so, you could use this to see what band disappears.
4. running beta actin will help you to determine that you have loaded protein (hopefully equally) between your samples, but it will not help you in terms of your other antibody. It will just tell you that you have protein on your blot. I would call the antibody company for suggestions.
I agree with yours, Marc's and Brandon's assessment in that a positive control is very important to validate the antibody.
Beta actin will help you to control the amount of loaded protein between your samples.
I recommend to repeat the blot, runing the beta actin and include the positive control recommended by the company.
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