Hi all. I need to evaluate Polyphenol oxidase (PPO) protein content in olive fruit extracts by Western blot. So I'm searching also a housekeeping protein in Olea europea for normalization. Some authors use ubiquitin2 gene for normalization in Real Time PCR experiments.
Do you known other proteins I can use to normalize my results?
Thank you very much for your answers.
@Luigi
Please excuse my lack of brevity. I am trying to be thorough at the outset of your research project. Hope the information below helps. Buona fortuna!
Instead of normalizing to a single housekeeping protein (HKP), have you considered normalizing to the total protein per lane? HKPs for Western Blot normalization need to be validated because their levels of expression may not be directly proportional to their corresponding mRNA transcripts. Total protein normalization will also yield more robust and comprehensive denominators to mitigate the effect of systematic variations, e.g., differential transfer of proteins from the separating gel onto the blotting membrane. Some major publishers such as the Journal of Biological Chemistry now prefer total protein normalization (see http://www.jbc.org/site/misc/ifora.xhtml#blots ). Also, total protein normalization can be less expensive than HKP normalization because fragile antibodies are not involved.
After doing a Bradford or BCA Assay to determine the protein concentration of your bulk sample, you have two options for total protein normalization, depending on the amount of sample remaining (be sure to pipette equal volumes of sample in each lane):
(A) If you have enough to prepare two or more gels in parallel, after electrophoresis one gel can be used for Coomassie staining (total protein denominators) while the other gel can be further processed for Western Blotting (target signal numerators). If you are detecting the Western blot signal by ECL on film or colorimetry, then you will have to use a scanner for densitometry. Below are published examples from plant biologists who used this approach to study olive-derived samples. For specific details and reprints, please contact the corresponding authors:
• "Isolation and molecular characterization of three acyl carrier protein genes in olive (Olea europaea L.)" (Italy, 2014)
http://link.springer.com/article/10.1007/s11295-014-0730-4
• "Cellular localization and levels of pectins and arabinogalactan proteins in olive (Olea europaea L.) pistil tissues during development: implications for pollen–pistil interaction" (Spain, 2012)
https://www.researchgate.net/profile/Agnieszka_Zienkiewicz/publication/232247500_Cellular_localization_and_levels_of_pectins_and_arabinogalactan_proteins_in_olive_Olea_europaea_L_pistil_tissues_during_development_Implications_for_pollen-pistil_interaction/links/0046351bb791521910000000.pdf
• "Characterization of a caleosin expressed during olive (Olea europaea L.) pollen ontogeny" (Spain, 2011)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180362/
• "Development of the cotyledon cells during olive (Olea europaea L.) in vitro seed germination and seedling growth" (Spain, 2011)
https://www.researchgate.net/profile/Agnieszka_Zienkiewicz/publication/49632905_Development_of_the_cotyledon_cells_during_olive_Olea_europaea_L_in_vitro_seed_germination_and_seedling_growth/links/02e7e51bb79bf5d742000000.pdf
(B) If you don't have enough to prepare two or more gels in parallel, a reversible membrane stain such as Ponceau S or REVERT can allow you to measure the total protein in each lane. The reversal ability helps to avoid interference in detecting your target. The choice of stain depends on your target detection chemistry. If you are using ECL, then more research has been done with Ponceau S. If you are using quantitative near-infrared fluorescence on an Odyssey imager, then REVERT is recommended.
Finally, here are some references from olive researchers who have used Western Blots to study your specified target (PPO) and other relevant proteins. These are not open-access, but perhaps the corresponding authors would be willing to share reprints (and technical suggestions) with you:
• "Phenol metabolism in the leaves of the olive tree (Olea europaea L.) cv. Picual, Verdial, Arbequina, and Frantoio during ripening" (Spain, 2010)
https://www.ncbi.nlm.nih.gov/pubmed/21047129
• "Polyphenol oxidase and its relationship with oleuropein concentration in fruits and leaves of olive (Olea europaea) cv. 'Picual' trees during fruit ripening" (Spain, 2008)
https://www.ncbi.nlm.nih.gov/pubmed/17938113
• "Photosynthetic activity during olive (Olea europaea) leaf development correlates with plastid biogenesis and Rubisco levels" (Israel, 2008)
https://www.ncbi.nlm.nih.gov/pubmed/18636989
Article Cellular localization and levels of pectins and arabinogalac...
Article Development of the cotyledon cells during olive (Olea europa...
Hello
I would also recommend normalising to total protein per lane. I think this could be the best way to do this, regardless of tissue. Have you considered purchasing some Ponceau stain? It is relatively cheap and essentially detects the total protein per lane after transfer to the membrane. You can then normalise to the total amount of protein.
http://www.sigmaaldrich.com/catalog/product/sial/p7170?lang=en®ion=AU
Good luck!
Hello Luigi
GAPDH, PP2A, EF1-alpha, OUB2, TIP2, alpha TUB, 60S RBP L18-3 are some of the common housekeeping protien that are widely used for expression studies. May i suggest to go for alpha TUB as it is a common housekeeping widely used for expression studies in both plant and animal cellular extracts
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis
- genetic techniques
- Sequencing