Dear all,
I collected roots, stems and leaves of pepper for analysis of enzymatic activity. I saw some people use liquid nitrogen to grind the plant samples before adding a buffer to extract proteins. However, some researchers do not use liquid nitrogen but they grind their samples directly in ice-cold buffer. So, can you please tell me about the advanges/disadvantages of each method? Thank you!
Dr. Hua, homogenization of tissue in solution often performed with cell lysis, you may grind and homogenized to physically break the robust cell. To prevent lysis, the tissue can be kept at temperature slightly above zero to prevent autolysis, and in an isotonic solution to prevent osmotic damage ( Chang et al. 1981). If freezing the tissue is possible, Cryohomogenization can be performed in the dry conditions, and it is a good method to choice. Cryohomogenization can be carried out using a super cooled mortar and pestle or tissue can be homogenized by crushing it into a fine power inside a clean plastic bag resting against a super cooled solid metal block ( Ziegler et al. 2013). You may also use chemical method for lysing cells without grinding ( Tsugama et al 2011). In this method plants are boiled for just 10 minutes in a solution containing Ca+ chelator and detergent. Cell extract prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method also applicable to genomic DNA extraction for PCR analysis. I think, in your case Cryohomogenization is the best.
Unlike animal cells, plant cells have cell walls comprised of hard substances like cellulose and lignin. Therefore, when you try to extract something like proteins/enzymes from plant tissues, it is hard to grind the tissue into a fine powder due to the presence of these substances which eventually leads to a poor extraction. Therefore, we add liquid nitrogen to make the plant tissue brittle which eventually allows you to grind the tissue into a fine powder. This allows you a better extraction. However, do not add the extraction buffer to the very cold tissue sample as the cold temperature will crystalize your buffer. Let the sample thaw a little bit and then add the extraction buffer gradually. Hope this will be helpful!
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