Cell Lysis Buffer for IP (Animal cells and plant cells)

Overview

Description

Cell Lysis Buffer for IP (Animal cells and plant cells)

Tested applications

IP

Properties

Components

pH 7.4; contains EDTA, 1% Triton X-100

Form

Liquid

Storage instruction

Store at +4°C.

Applications

Highlights

• Optimized for compatibility with immunoprecipitation and pull-down assays

• Compatible with protein assays, reporter assays and immunoassay procedures

• Ready-made formula is effective for extracting cytoplasmic, membrane and nuclear proteins

• Does not liberate genomic DNA which can cause high sample viscosity

Protocols

Procedure for Lysing Cell Monolayer (Adherent) Cultures

1. Carefully remove culture medium from cells. Wash the cells once with ice cold phosphate-buffered saline.
 

2. Add ice cold lysis buffer to the cells according to the table below and incubate on ice for 5-10 minutes with periodic mixing. 

Size	Volume of Buffer
100 mm dish	500-1,000 μL
60 mm dish	250-500 μL
6-well plate	200-400 μL per well
24-well plate	100-200 μL per well

3. Transfer the lysate to a microcentrifuge tube and centrifuge at ~13,000 × g for 10 minutes to pellet the cell debris at 4°C.
 

4. Transfer supernatant to a new tube for protein concentration determination and further analysis. 

Procedure for Lysing Cell Suspension Cultures

1. Centrifuge the cell suspension at 1,000 × g for 5 minutes to pellet the cells. Discard the supernatant.
 

2. Wash the cells once with ice cold PBS. Centrifuge at 1,000 × g for 5 minutes to pellet cells.
 

3. Add ice cold IP Lysis Buffer to the cell pellet. Use 500 μL of lysis buffer per 50 mg of wet cell pellet (10:1 v/w).
 

Note: If using a large amount of cells, first add 10% of the final volume of lysis buffer to the pellet and pipette the mixture up and down to mix. Add the remaining volume of lysis buffer to the cell suspension.

4. Incubate lysate on ice for 5-10 minutes with periodic mixing. Remove cell debris by centrifugation at ~13,000 × g for 10 minutes at 4°C.
 

5. Transfer supernatant to a new tube for protein concentration determination and further analysis. 

Troubleshooting Guide

1. all reagents and lysates must be kept cold
2. Cell Lysis Buffer can be used for lysis of tissue samples, although a homogenization step is recommended after adding lysis buffer. Extract the tissue at a ratio of 100 mg of tissue to 1 ml of buffer. Sonication of the tissue lysate is also required.
3. The buffer does not contain protease or phosphatase inhibitors; Protease Inhibitor Cocktail or Phosphatase Inhibitor Cocktail should be added just before use to prevent proteolysis and maintain phosphorylation of proteins.