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Upstate - Procedures and Protocols - Preparation of Cell Lysate
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Upstate Cell Signaling Symposium
 
 
The first annual Upstate Cell Signalling Symposium will take place in Dundee, Scotland (UK) from 6 - 9th June 2004 and is entitled:

Regulation and Therapeutic Potential of the PI 3-kinase/PKB Signalling Pathway.

The symposium is co-sponsored by Sir Philip Cohen and Prof. Dario Alessi (The MRC Protein Phosphorylation Unit) in conjunction with Upstate.

Participation is strictly limited to 300 delegates, so early registration is recommended.



Preparation of Cell Lysate


1. Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS. Wash non-adherent cells in PBS and centrifuge at 800 to 1000 rpm in a table-top centrifuge for 5 minutes to pellet the cells.

2. Add ice-cold modified RIPA buffer to cells (1 ml per 107 cells/100 mm dish/150 cm2 flask; 0.5 ml per 5 x 106 cells/60 mm dish/75 cm2 flask).

3. Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell scraper that has been cooled in ice-cold distilled water. Transfer the cell suspension into a centrifuge tube. Gently rock the suspension on either a rocker or an orbital shaker in the cold room for 15 minutes to lyse cells.

4. Centrifuge the lysate at 14,000 x g in a precooled centrifuge for 15 minutes. Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet.

5. Dilute the cell lysate at least 1 : 10 before determining the protein concentration because of the interference of the detergents in the lysis buffer with the Coomassie-based reagent. At this step, the sample can be divided into aliquots and stored at -20íC for up to a month.

TIP: When working with large volumes of non-adherent cells, the cells may not be cooled quickly enough to maintain the activity of the protein being studied. In this case, pour the cell suspension into a mixture of an equal mass of 2 x PBS and ice, then collect the cells by centrifugation and perform the lysis as described above.

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Preparation of Modifed Radioimmunoprecipitation (RIPA) Buffer

Activation of Sodium Orthovanadate

Preparation of Cell Lysate

Western Immunoblotting of Proteins

Troubleshooting Tips for Western Immunoblotting

Immunoprecipitation of Proteins

Immunoprecipitation / Kinase Assay

Troubleshooting Tips for Immunoprecipitation / Kinase Assays

Assay for Immunoprecipitated Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro)

Phospholipid Vesicle Preparation

Chromatin Immunoprecipitation (ChIPs) Assay Kit

Forskolin Treatment of Cells

Preparation of Brain Cytosol

Immunohistochemistry with Fixed Paraffin-Embedded Tissue Sections

Tips for Immunohistochemistry with Fixed Paraffin-Embedded Tissues

Immunohistochemical Methods to Detect Nitrotyrosine

Immunocytochemistry with Adherent Cells

Differentiation of Stages in the Cell Cycle

Elution of GST-Fusion Protein from Glutathione Agarose

Purification of Antiserum or Ascites by Protein A/G Chromatography

Molecular Weights of Some Common Proteins

Transient Transfection of Cos-1 Cells

The Genetic Code
 
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