Dear colleagues,
We are having an issue with the Anti-4-Hydroxynonenal Antibody (AB5605 from Merck). We've been using this antibody for the Slot Blot technique, but it seems to react to just H2O samples (negative control). At first we thought it was the antibody that was contaminated and ordered a new aliquot and the issue persists. Interestingly, the staining seems to be more intense with H2O.
The slot blot procol is performed as follow:
1. Dilution of 5ug protein extracted with RIPA buffer in Mili-Q H2O;
2. PVDF membranes are activated 1min in methanol and 5 minutes Mili-Q H2O;
3. Transference of protein or just H2O (negative control) to PVDF membranes by Slot Blot;
4. Membranes are blocked in TBS-T 5% non-fat milk for 1h at RT;
5. Membranes are washed in TBS-T 3 times for 5min each;
6. Membranes are incubated with Anti-4-Hydroxynonenal Antibody (1:5000 in TBS-T 1% non fat milk) overnight at 4ºC;
6. Membranes are washed in TBS-T 3 times for 5min each;
7. Membranes are incubated with anti-goat HRP (1:5000 in TBS-T 1% non fat milk) for 1h30 at RT;
8. Incubation with ECL.
The problem only exists with this antibody, thus we concluded that the problem does not concern the slot blot technique.
I hope you can help me.
Best regards,
David
Your only option is to systematically try all components of your assay to identify the culprit. I once had a problem with detergents in the sample, that unspecifically bound primary antibody. Also, try the concentration of the secondary antibody.
Looks like your antibody is binding to your blocking agent. Based on the description below, from the SigmaAldrich website this is quite possible with this antibody. I would try a different blocking agent that does not have lipids. I know you said you use non-fat dry milk, but the milk starts out with lots of lipids and the reactions that would provide a target for your antibody may have formed long before the milk was de-lipidated. I would try Albumin, or there are some Fish based blocking agents that I have used for problematic antibodies.
Specificity
Specific for 4-Hydroxynonenal (4-HNE).
The HNE moiety recognized by this AB5605 antibody is a biochemical modification independent of species. The anit-4-HNE antibody should have wide species reactivity accordingly.
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