Homogenization

Buffers:
#1 100 mM potassium phosphate, pH 7.4
150 mM KCl
1 mM EDTA

#2 100 mM tetrasodium pyrophosphate, pH 7.4
1 mM EDTA

#3 100 mM potassium phosphate, pH 7.4 (Suspension Buffer)
1 mM EDTA
20% glycerol

Procedure:
1. Wash chopped tissue several times with ice cold saline solution.
2. Weigh tissue and add 4x volume of Buffer #1
3. Homogenize in Waring blender using two 45 second blends
4. Centrifuge mixture for 35 min at 9,000 g in a Sorvall centrifuge (GSA rotor; low speed centrifuge)
5. Discard pellet
6. Filter supernatant through 4 layers of cheesecloth
7. Spin filtered supernatant at 105,000 g for 75 minutes in a T35 rotor. (ultracentrifuge)
8. Suspend pellets in a minimal volume of Buffer #2 using a Potter-Elvehjem homogenizer.
9. Spin at 105,000 g for 75 minutes in a T35 rotor. (ultracentrifuge)
10. Suspend pellets in Buffer #3 using a Potter-Elvehjem homogenizer. Use ~1 mL of buffer / 2 grams of tissue.
11. Store in 1.0 mL aliquots in –70ºC.

This general protocol is is intended for use as a reference as a courtesy to our customers. Optimal concentrations, experimental conditions and experimental processes are to be determined by the individual user. No guarantee of performance using the above procedure is expressed or implied. Molecular Innovations does not currently perform homogenization of tissues in-house and can not provide further technical support for this application.

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