Mammalian Cell Lysis Reagent (提取的蛋白具有活性）

Mammalian Cell Lysis Reagent

Introduction

The Buffer is a mammalian whole cell lysis buffer based on a modified RIPA buffer formulation without SDS. This moderate strength lysis buffer is able to effectively solubilize cellular proteins but does not liberate genomic DNA or disrupt protein complexes like RIPA buffer. The Buffer is especially formulated for pulldown and immunoprecipitation assays and as a wash buffer for affinity resins. This lysis buffer is also compatible with many other applications, including protein assays, protein purification and immunoassays (e.g., ELISA, Western blot). 

Important Product Information 

• The Buffer does not contain protease or phosphatase inhibitors. If desired, add Protease and Phosphatase Inhibitor Cocktail immediately before use.

• The Buffer has been tested on representative cell types including the following cell lines: HeLa, Jurkat, A431, A549, MOPC, NIH 3T3, HepG2, COS-7 and U2OS. Typically, 106 HeLa cells yield ~10 mg of cell pellet and ~3  μg/ μl (or 300 μg) using 100 μl of lysis buffer.

• To use the Buffer with the Active GTPase Pull-Down and Detection Kits, add MgCl2 to a final concentration of 6 mM to neutralize EDTA. Some reporter assays, such as luciferase, might require additional cofactors.

• The Buffer is compatible with the BCA Protein Assay .

Procedure for Lysing Cell Monolayer (Adherent) Cultures  

1. Carefully remove culture medium from cells. Wash the cells once with ice cold phos phate-buffered saline (PBS).

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

 Plate Size/Surface Area Volume of The Buffer

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 the 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 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.

Procedure for Tissue Lysis

1. If desired, add protease inhibitors to the Buffer just before use.  

2. Weigh tissue samples. Use a ratio of ~1 g of tissue to 20mL the Buffer. Use a smaller volume of the Buffer if a more concentrated protein extract is required.  （Example :Use 1ml of lysis buffer per 50mg of tissue ).

3. Add the appropriate amount of the Buffer to the tissue sample and homogenize.

4. Pipette the mixture to a new tube, Shake mixture gently for 30 minutes on ice.

5. Centrifuge the sample at 14,000 × g for 15 minutes to pellet cell/tissue debris.  

6. Collect supernatant and continue with downstream analysis or further purification.

Origin:  Pioneer Biotechnology,  Inc of China

Distributor: Pioneer Biotechnolgy,Inc

Contact:  Tel 029-88237772 or 13484545009  Fax 029-88237772

  E-mail : xfyangbio@163.com

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