Question

I need to extract total protein from 2mm skin biopsies for western blots. I was thinking about miRNeasy Micro Kit (50) Cat. while it's ok for total RNA extraction, I am not sure if it is still fine for proteins. There's a trizol (quiazol) step that provides you with organic fraction that suppose to contain protein fraction, but I am still not sure if it's a best way. There's also a so-calledUrea Sample Buffer (USB), that contains urea, 20% SDS, glycerol, Tris and Pyronin Y - but I am not sure it's a good choice since the percentage of SDS is high and it will be a disaster if I will try to disrupt my tissue. Or, maybe there's a better solution or kit for that purpose?

Answer 1

For protein extraction from 2mm skin biopsies, using a method that involves re-extraction and precipitation with buffers may provide a cleaner protein sample, rather than relying on RNA extraction kits. Direct tissue homogenization and various solubilization buffers, possibly using different combinations of urea, thiourea, CHAPS, and DTT, are recommended to increase the quality of the extracted proteins for western blots.

Step-by-step explanation:

Protein Extraction Methods for Western Blots

For efficient protein extraction from 2mm skin biopsies intended for western blotting, the choice of the extraction method is crucial to obtain high-quality proteins. Using kits like the miRNeasy Micro Kit, which is typically used for total RNA extraction, may not be suitable since the organic fraction obtained from a trizol step may not yield the best quality of protein. However, a method involving re-extraction and precipitation with buffers may provide a cleaner protein sample. The urea sample buffer (USB) that contains high percentages of urea and SDS could potentially disrupt the tissue, so an alternative protocol that ensures the integrity of the proteins while effectively solubilizing them is recommended.

Within scientific literature, various protein extraction methods have been evaluated, such as phenol extraction, TCA/acetone precipitation, and direct tissue homogenization in protein solubilization buffers. These methods have been used to generate quality proteome maps in soybean leaf and flower proteomes, with modifications used to optimize protein pellet solubilization buffer. For instance, buffers with combinations of urea, thiourea, CHAPS, DTT and more are tested for efficacy in extracting proteins without the interference of secondary metabolites or storage proteins, which can be problematic in plants like soybeans.

Moreover, the process of protein extraction may include the use of lysis buffers, sonication, centrifugation, and other steps for sample preparation. It is important to consider reagents like DTT and iodoacetamide for protein reduction and alkylation, and to use appropriate mass spectrometry techniques to analyze the peptides after digestion.