Our lab is looking into Western Blot alternatives to the classic membrane method, and are developing a protocol for mouse brain tissue preparation. We have whole tissue that we are determining the best way to refine before using in the protocols. Many tissue prep protocols have used homogenization alone, and other use sonication devices. What is the best/ most cost effective method here? What kind of sonication device would be necessary (power, wattage, etc.) to complete such a task without going overboard? These are very expensive devices, so we don't want to have to purchase a more expensive item than is necessary.
More sensitive tissue can be lysed with sonication rather homogenization.
1. Sonication involves more power which lyse the cell/tissue prep efficiently within shorter time. Since there are two types of sonicators (Bath and probe) available in the market, I believe you are talking about probe sonicator.
2. The working sample volume is much less in sonication (Considering availability of probe sonicator with microprobe attached). Since homogenization is a rubbing and friction-based technique the working sample volume is usually large.
3. Sonication is much more efficient than homogenization as sonication disrupts DNA integrity which makes nuclear proteins easily extractable but in other words that makes DNA vulnarable for disintegration and samples may not be good for nucleic acid analysis.
For whole tissue prep, homogenization alone will work and you need to centrifuge the sample to obtain specific pools (cytosolic, nuclear, mitochondrial or lysosomal...) to enrich the protein for analysis.
Starting with big volume of tissue size (Kidney, heart, lung etc), after mincing the samples into very small pieces with razor blades I use IKA homogenizar (Power setting 2-4, samples kept in ice) with lysis buffer (RIPA or hypotonic lysis buffer containing protease and phosphatase inhibitor cocktail) for a total of 1-2 min, with 15 s run and 15 s pause to limit sample heating and visually inspect for big chunks.
One a moderately clear lysate is obtained I use Misonix 3000 ultrasonicator (Power setting 3, 6W) a total run of 1 min (15s run and 10s pause), saples kept in ice.
Then I do differential centrifugation steps to get the target fraction enriched with proteins of interest.
As a ready reference I am sharing photos of these two instruments along with a relevant paper where the procedure has been described.
As an alternative to membrane-based classical WB protocol you can think of trying In Cell and On cell westerns when you work with cultured cells in well plate format.
Do I prefer sonication of spleen and liver samples for DNA study for Leishmaniasis?
If I want to prepare Emulsion (Essential oil and Gum Arabic) for Spray drying and microencapsulating the EO, what should i use Sonicater or Homogenizer.
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