Homogenization and Solubilization of RASSLs

1. Place 1 ml of homogenization buffer in a round bottom 14 ml tube.

2. Transfer the frozen tissue into the 14 ml tube. Homogenize using a homogenizer at max speed until tissue is fully ground (about 30-60 seconds).

3. Pipet the homogenate to an eppendorf tube and immediately place it on ice.

4. If the homogenate is too viscous, sonicate for 3-5 seconds.

5. Normalize samples by tissue weight and use approximately 10-50 mg of tissue for solublization.

6. Solublize each sample in an eppendorf tube using the following conditions:

• 1x SSB
• 1x Triton-X 100
• 1x Complete^TM Cocktail (Protease Inhibitor, Boehringer #1697498)

Example:

x ml of homogenate (~10-50 mg tissue)

100 ml 10x SSB

100 ml 10% Triton-X 100

100 ml 10x Complete ^TM cocktail

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1000 ml (adjust volume using ddH₂0)

7. Incubate for 30 min at 4°C on a rotator.

8. Spin for 5 min at 4°C at max speed.

9. Transfer supernatant to a new eppendorf tube.

 

Buffers Homogenization Buffer

• 50mM Tris-HCl, pH 7.4
• 1x Complete^TM Cocktail
• *1mM DTT
• *1mM PMSF
• in dd H₂0

*unstable in water, therefore, use buffer within 30 min after addition of PMSF, DTT

10x Complete^TM Cocktail

• Dissolve 1 tablet (Cat# 1697498) in 5 ml H₂O

10x SSB

• 100 mM Tris-HCl, pH 7.4
• 1.5 M NaCl

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