I am working on determining malondialdehyde (MDA) content in Penstemon leaves following protocol: http://www.bio-protocol.org/e1913. After crude enzyme extraction in buffer I was going to figure out protein concentration.
But, I obtained absorbance like 4 or higher at A280 and A260. And even if diluted many times the absorbance is still higher.
It would be great help if anyone can provide me suggestion.
Thanks!
The absorbance at 280 nm is not protein-specific. Your sample may contain non-protein substances from the leaf extract (e.g. pigments) that are causing a high absorbance. Turbidity (cloudiness) of the sample will also cause a high apparent absorbance, which is especially strong in the UV. I suggest you try using a protein-specific protein assay, such as the Bradford or BCA or Lowry assay. If those assays also go off-scale, you should try diluting the sample more.
Whatever extract you got from leaf, first you cheak for protein identification by performing Biuret test, showing Violet colored ppt to your sample. If this happen then you must go to total protein determination present in leaf extract by using Lowrys method at 280 nm. You also called your sample by Biuret estimation at 620 or 680 nm . In this estimation no need to dilution to your original extract.
- Badges
- Science topic
- Similar topics
- Filing