Is there an antibody that works well in a western to detect proteins that have been conjugated to malondialdehyde (MDA), the reactive aldehyde lipoxidation product? Could you recommend a good commercially available antibody? Thanks!
I have worked on aldehyde-modified proteins for many years and have found that most of the commercial anti-MDA antisera are not very good. I raised my own and have found them to work fine. Rather than using anti-MDA-modified protein antisera you could react your proteins with dinitrophenylhydrazine to produce DNP-hydrazones. These can then be picked up with COMMERCIAL anti-DNP-hydrazine antisera which work well.
Thanks, Simon! Forgive my ignorance, but is this the same as the so-called "oxyblot"? Do you have a reference to recomend for the methodology/protocol?
Yes this is similar to the oxyblot.
Here is my protocol with references.
DETERMINATION OF PROTEIN OXIDATION BY SLOT BLOTTING
Oxidation and nitration of proteins can be assessed by measuring protein carbonyl and nitrotyrosine formation. Carbonylation and nitrotyrosination of proteins can be determined in a similar manner apart from differences in the primary antibody and buffer used [1, 2]. This also works for Western blotting and ELISA.
REAGENTS REQUIRED
For carbonyl content: 2N HCl; 2, 4-dinitrophenylhydrazine (2, 4-DNP) at 100 mg/ml in 2N HCl; 5% (w/v) BSA in PBS-Tween 20
For nitrotyrosine content: general reagents
TBS/milk/Tween
PROTOCOL
1. Blot 1 mg of protein onto a PVDF membrane.
2. For carbonyl content: incubate in 2N HCl and 2, 4-DNP (100 mg/ml in 2N HCl) for 5 min before blocking with 5% BSA in PBS-Tween 20. Probe blots with anti-DNP antibody (1:25,000 dilution; Molecular Probes, Eugene, OR) for 60 min at RT. Wash and then incubate with HRP -conjugated secondary antibody (1:4000) for 160 min at RT.
For nitrotyrosine content: block with 5% BSA in PBS-Tween 20. Probe blots with anti-nitrotyrosine antibody (Upstate Biotechnology, Charlottesville, VA) for 60 min at RT. Wash and then incubate with HRP -conjugated secondary antibody (1:4,000) for 60 min at RT.
3. Wash membranes 6 times (5 min) in TBS/milk/Tween, add chemiluminescence reagent and expose to film.
4. Quantify by laser densitometer.
[1] Robinson, C. E., Keshavarzian, A., Pasco, D. S., Frommel, T. O., Winship, D. H. and Holmes, E. W. (1999). Determination of protein carbonyl groups by immunoblotting. Analytical Biochemistry 266, 48-57.
[2] Banan, A., Choudhary, S., Zhang, Y., Fields, J. Z. and Keshavarzian, A. (1999). Ethanol-induced barrier dysfunction and its prevention by growth factors in human intestinal monolayers: Evidence for oxidative and cytoskeletal mechanisms. Journal of Pharmacology and Experimental Therapeutics 291, 1075-1085.
Hi Beverley,
We just launched two new monoclonal antibodies that bind to malondialdehyde (MDA) conjugated proteins. They have both been validated for use in western blot (among other applications). Here's the info:
Malondialdehyde Antibody, Clone 6H6 Catalog No. SMC-514
- http://www.stressmarq.com/products/antibodies/malondialdehyde-antibody-smc-514/
Malondialdehyde Antibody, Clone 11E3 Catalog No. SMC-515
- http://www.stressmarq.com/products/antibodies/malondialdehyde-antibody-smc-515/
We also offer free antibody samples, where you can receive a 12 ug vial to test before you buy a full size vial. Here's the info for the free antibody samples:
- http://www.stressmarq.com/free-antibody-samples/
Hope this helps!
Leslie
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