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Cell Lysis Buffer (10X) #9803

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Description

Cell Lysis Buffer is used to lyse cells under nondenaturing conditions.

Directions for Use

1. If buffer will be continually used, it is recommended that the 10x buffer be kept at 4°C for 1-2 weeks. For longer periods of time, buffer should be stored at -20°C. Aliquotting of 10x buffer is recommended if many small experiments are to be performed.
2. Thaw 10x buffer at 24-30°C, mixing end-over-end.
3. Dilute 10x buffer in Milli-Q water or water of equivalent quality.
4. Chill 1x buffer on ice and add PMSF just prior to use.

Lysis:

For lysis of adherent cells, we recommend the following: (all reagents and lysates must be kept cold).

1. Treat cells as desired.
2. Wash plate with PBS to remove residual media.
3. Add 400 ul of 1x lysis buffer/10 cm dish.
4. Incubate plate on ice for 5 minutes.
5. Scrape cells.
6. Sonicate briefly.
7. Centrifuge extract for 10 minutes at 14,000 x g in a cold microfuge.
8. Remove supernatant for use.

Additional notes:

1. For non-adherent cells, judgment must be used as to the volume of lysis buffer to add. Addition of volume equal to cell pellet volume is generally appropriate.
2. The CST 10x buffer can be used for lysis of tissue samples, although a homogenization step is recommended after adding lysis buffer.
3. Additional protease inhibitors can be added to the 1x lysis buffer without any difficulties.

Solutions and Reagents

• 20 mM Tris-HCl (pH 7.5)
• 150 mM NaCl
• 1 mM Na₂EDTA
• 1 mM EGTA
• 1% Triton
• 2.5 mM sodium pyrophosphate
• 1 mM beta-glycerophosphate
• 1 mM Na₃VO₄
• 1 µg/ml leupeptin

Note: CST recommends adding 1 mM PMSF immediately before use.

Application References

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Companion Products

• 9802 Kinase Buffer (10X)
• 9806 RIPA Buffer (10X)