实验概要

Primary tissues  are valuable tools for the study of intracellular and extracellular  markers which characterize disease states. We have developed a protocol  for rapid isolation of cytokines and signaling molecules from intact  tissue. This method is for total protein extraction and makes use of a  non-abrasive tissue extraction reagent. Tissue samples as little as 10  mg may be extracted using this protocol. This method is sensitive and  allows for the detection of disease-associated fluctuations of  biomarkers. This is an effective system for the extraction of proteins  from a variety of tissue types. Heart, lung, kidney, spleen, brain,  liver, thymus, and smooth muscle tissues have all been successfully  extracted with this protocol. The prepared extracts are compatible with a  variety of immunoassays, such as ELISA and Luminex®, as well as with  all conventional protein assays. Our extraction method is inexpensive,  versatile, and can be completed in less than 15 minutes. Invitrogen™  offers a Cell Extraction Buffer for total protein extractions from  various tissue sample types.

主要试剂

Prepare 1x Cell Extraction Buffer using the following formulation:

Cell extraction buffer formulation

* 100 mM Tris, pH 7.4
* 2 mM Na₃VO₄
* 100 mM NaCl
* 1% Triton X-100
* 1 mM EDTA
* 10% glycerol
* 1 mM EGTA
* 0.1% SDS
* 1 mM NaF
* 0.5% deoxycholate
* 20 mM Na₄P₂O₇
This Cell Extraction Buffer is available from Invitrogen (Cat. no. FNN0011).

This  Cell Extraction Buffer may be apportioned into 1x aliquots in  microcentrifuge tubes and stored at –20°C until ready for use. Thaw on  ice prior to extracting cells.

Additional Reagents Needed:

1 mM PMSF
Protease Inhibitor Cocktail, Sigma (Cat. no. P-2714)

This Cell Extraction Buffer must be supplemented with 1 mM PMSF (not  included) and Protease Inhibitor Cocktail (not included) just prior to  use to make Complete Cell Extraction Buffer. Addition of the Protease  Inhibitor Cocktail and PMSF is necessary to inhibit proteolysis in cell  extracts. For the PMSF addition, we recommend making a 0.3 M stock in  DMSO, and adding sufficient volume for a final concentration of 1 mM  (i.e., 17 μl per 5 ml Cell Extraction Buffer). PMSF is very unstable and  must be added just prior to use, even if added previously. For the  Protease Inhibitor Cocktail addition, we recommend Sigma (Cat. no.  P-2714), reconstituted according to the manufacturer’s instructions, and  adding 250 μl per 5 ml Cell Extraction Buffer. The stability of  protease inhibitor-supplemented Cell Extraction Buffer is 24 hours at  4°C.

实验步骤

Tissue Culture Sample Protocol

1.      Following the end of the desired cell culture time, pipette medium into a microcentrifuge tube and

2.      immediately put on ice.

3.      Centrifuge at 1,400 rpm for 1 minute.

4.      Remove supernatant fluid and aliquot into a clean microcentrifuge tube.

5.      Store at –80°C until ready for use in ELISA.

Cell Extraction Protocol

Processing Cells

This  method can be used to produce relatively large quantities of cell  extracts with each of the stimulation regimes studied. The wash steps  included in this procedure help to minimize medium components in the  cell extracts.

1.       Estimate cell density: Suspension Cells: Enumerate suspension cells by  counting in a hemacytometer. Adherent Cells: Estimate cell density by  visual inspection under a microscope. Confluence levels of 70–80% are  found to be optimal for many signal transduction studies.

2.      Stimulate cells as desired.

3.       Transfer the cells into clean 15 ml conical tubes: Suspension Cells:  Aliquot the desired number of cells in medium into clean 15 ml conical  tubes. Adherent Cells: Remove the cells from their vessel by scraping.  Transfer the medium containing the detached cells into clean 15 ml  conical tubes.

4.      Collect the cells by centrifugation at 300 x g for 7 minutes.

5.      Aspirate the medium.

6.      Resuspend the pellet in ice-cold PBS.

7.      Collect the cells by centrifugation at 300 x g for 7 minutes at 4°C.

8.      Aspirate the PBS.

9.       Lyse the cells by pipetting Complete Cell Extraction Buffer into each  tube. We recommend using 1 ml of Complete Cell Extraction Buffer per 10⁸ cells. It is important to note that this value may require optimization for each specific application.

10.  Transfer the lysates to clean microcentrifuge tubes.

11.  Vortex the mixture, then incubate the mixture on ice for 30 minutes, with occasional vortexing.

12.  Clarify the lysates by centrifugation at 14,000 rpm (13,000 x g) at 4°C for 10 minutes.

13.  Transfer the clarified cell extracts to clean microcentrifuge tubes.

14.   The clarified cell extracts should be stored at –80°C until ready for  analysis. Avoid repeated freeze-thaw cycles. In preparation for  performing the assay, allow the samples to thaw on ice. Mix well prior  to analysis.

15.   Determine protein concentration using a suitable method, such as the  Quant-iT™ Protein Quantitation Kit (Cat. no. Q33210). Cell extracts  prepared by this method routinely have a protein concentration between 1  and 10 mg/ml.

16.   Certain analytes require a sample treatment step. Please refer to the  analyte specific protocol for details on sample treatment  recommendations.

17.   All cell extracts require dilution by a factor of at least 1:10 in  Standard Diluent Buffer before analysis with Invitrogen™ kits.