29 Protein extraction method for adherent cell cultures IFR UREAD

NuGO approved proteomics keywords: 2-D gel electrophoresis, protein extraction, cell lines

SopID: 29- IFR/UREAD

When citing this SOP you should acknowledge both NuGO and the appropriate NuGO partner institution that has made the SOP available. Please use a form of words such as: We used the NuGO Standard Operating Procedure (SOP) number 29 produced by the University of Reading and IFR Norwich
Dr Abigael Polley, IFR, Norwich, UK Dr Anne M Minihane, Hugh Sinclair Human Nutrition Group, School of Food Biosciences, University of Reading, UK

Details of the SOP are available via the web link: http://www.nugo.org/frames.asp?actionID=28250&action=loginFromPP
Backgrounds
29This assay is modified from the BioRad ReadyPrep Sequential Extraction Kit (catalogue #1632100). Reagent 1 (40mM Tris) extracts the most soluble proteins, e.g. cytosolic proteins, and is modified to contain protease inhibitors, DNase and RNase to protect the sample from degradation. Additional protein fractions can be obtained using Reagents 2 and 3.

Overview Materials
Amount 40mM 2.5g/ml 5 units/ml 5g/ml 3mM Name Trizma base Protease inhibitor cocktail DNase I (RNase-free) RNase A MgCl2 Supplier Sigma Sigma Promega Sigma Sigma Catalogue Number T-6066 P-8340 M6101 R-6513 M-8266 Further information
This is the basic Reagent 1 from the BioRad kit

Main Procedures
1. This protocol is based on 25cm2 flask area. Adapt the volumes accordingly.

2. Wash cells. 3. Lyse cells in Sample Buffer by vortexing and sonication. 4. Centrifuge to obtain supernatant. 5. Determine protein concentration.
6. Freeze and store at -80C.

Sub Procedures 1. Prepare Trizma base in 18.2 water and freeze aliquots at -20C. Label as Reagent 1. This
can be stored for up to 6 months.

2. On the morning of extraction thaw Reagent 1 and keep on ice. Modify with the additional
chemicals from the table above. Label as Sample Buffer.

3. Treating each flask individually - aspirate the medium from a flask of confluent cells and wash

the cell layer washed twice with 2 ml 1xPBS (Phosphate Buffered Saline solution, Sigma, P4417).

4. Immediately aspirate the PBS and wash the cells with 2 ml Reagent 1 (NB no additions!)
5. Immediately aspirate Reagent 1 and add 1.25ml Sample Buffer to the cell monolayer. 6. Incubate the flask at room temperature for 5 minutes, remove the cell debris using a cell scraper and transfer the solution to a 1.5ml micro-centrifuge tube (Eppendorf, catalogue #: 0030120086).

7. Leave tube on ice until all flasks have been harvested.

8. Vortex tubes for 1 minute to mix thoroughly and sonicate in an iced waterbath for 10 minutes to completely lyse the cells.

9. Centrifuge (16,000 rcf for 5 minutes @ 4C) to obtain a pellet. Keep tubes on ice at all other
times.

10. Carefully transfer the supernatant with a fine tip pasette (Alpha Laboratires, catalogue #:
LW4060) and put into a labelled 0.5ml micro-centrifuge tube (Eppendorf, catalogue #: 0030121023). Keep on ice.

11. If less soluble fractions are required, follow standard issued protocol from BioRad
ReadyPrep Sequential Extraction Kit using Reagents 2 and 3.

12. Determine the protein concentration of the Extract using Amersham 2-D Quant Kit as per
standard issued protocol (GE Healthcare, Ettan Sample Preparation Kits and Reagents, catalogue #: 80-6483-56). 13. Store samples at -80C until required in 1st dimension run.

Safety
Users must comply with COSHH and local safety regulations.