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539720 ProteoExtract® Tissue Dissociation Buffer Kit

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      Overview

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      Description
      Overview

      This product has been discontinued.



      The ProteoExtract® Tissue Dissociation Buffer Kit is a tool for isolation of viable cells from fresh tissue. The isolated cells can then be used for total, subcellular, membrane, or other form of proteome extractions. This kit serves as an accessory product for the following kits: ProteoExtract® Subcellular Proteome Extraction Kit (Cat. No.: 539790), ProteoExtract® Subcellular Proteome Extraction Kit, Mini (Cat. No.: 539791), ProteoExtract® Native Membrane Protein Extraction Kit (Cat. No.: 444810).
      Catalogue Number539720
      Brand Family Calbiochem®
      Application Data

      Tissue dissociation of rat pancreatic tissue and human pancreatic tissue was carried out according to the protocol outlined above. 1.3 x 107 dissociated cells were extracted using the ProteoExtract® Subcellular Proteome Extraction Kit protocol (Cat. No. 539790), incorporating the protocol changes as outlined above. Proteins from the cytosolic fraction, the membrane fraction, the nuclear fraction, and the cytoskeletal fraction were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to the indicated markers.
      Materials Required but Not Delivered• Collagenase: the collagenase should efficiently and specifically dissociate the tissue type of choice. Based on internal testing, a minimum of 10,000 units (~8-10 mg) collagenase is recommended per gram of tissue. The exact amount will depend on the specific enzyme and type of tissue. The list of enzymes below represents possible options used successfully with this kit. Please always refer to the manufacturer's recommendations and specifications for use.

      Table 1: Collagenases Used With Various Tissues


      • Kit for downstream processing (optional; e.g. ProteoExtract® Subcellular Proteome Extraction Kit, Cat. No. 539790, ProteoExtract® Subcellular Proteome Extraction Kit Mini, Cat. No. 539791, or ProteoExtract® Native Membrane Protein Extraction Kit, Cat. No. 444810)
      • Hemocytometer
      • Phosphate Buffered Saline (PBS)
      • Pasteur or micropipet
      • Microscope
      • Trypan blue (e.g. 0.18% in PBS)
      • 15 ml conical centrifuge tubes
      • Thermo mixer
      • Sieve
      • Scalpel or scissors
      • Refrigerated centrifuge (minimum rcf = 350 g)
      • Petri dish
      • 37°C incubator with gentle agitation
      References
      Product Information
      Form10 Dissociation reactions, 1 g each
      Kit contains10X Tissue Dissociation Buffer, Protease Inhibitor Cocktail, Tissue Dissociation Buffer Additive and a user protocol.
      Quality LevelMQ400
      Applications
      Biological Information
      Sample TypeFresh tissue samples from eukaryotic organisms
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® ProteoExtract® Tissue Dissociation Buffer Kit is a tool for isolation of viable cells from fresh tissue for the purpose of protein extraction. It is an accessory product for the following kits:

      • ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK, Cat. No. 539790)
      • ProteoExtract® Subcellular Proteome Extraction Kit, Mini (S-PEK Mini, Cat. No. 539791)
      • ProteoExtract® Native Membrane Protein Extraction Kit (M-PEK, Cat. No. 444810).

      With the exception of the collagenase enzyme the set contains all reagents required for efficient dissociation of tissue samples. The goal of the cell isolation procedure is to maximize the yield of viable dissociated cells, so the end-user must select the collagenase appropriate for each tissue with respect to the type of tissue, the species of origin, and the age of the animal. A list of enzymes is provided below in Materials Required but Not Provided.
      Storage and Shipping Information
      Ship Code Ambient Temperature Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C. Following reconstitution, all components are stable for a minimum of 2 days at 4°C or for up to 2 months at -20°C. Avoid freeze/thaw cycles of reconstituted solutions.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit contains10X Tissue Dissociation Buffer, Protease Inhibitor Cocktail, Tissue Dissociation Buffer Additive and a user protocol.
      Specifications
      Global Trade Item Number
      Catalog Number GTIN
      539720 0

      Documentation

      ProteoExtract® Tissue Dissociation Buffer Kit Certificates of Analysis

      TitleLot Number
      539720
      User Protocol

      Revision17-September-2007 RFH
      Form10 Dissociation reactions, 1 g each
      StorageUpon arrival store the entire contents of the kit at 4°C. Following reconstitution, all components are stable for a minimum of 2 days at 4°C or for up to 2 months at -20°C. Avoid freeze/thaw cycles of reconstituted solutions.
      Intended useThe Calbiochem® ProteoExtract® Tissue Dissociation Buffer Kit is a tool for isolation of viable cells from fresh tissue for the purpose of protein extraction. It is an accessory product for the following kits:

      • ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK, Cat. No. 539790)
      • ProteoExtract® Subcellular Proteome Extraction Kit, Mini (S-PEK Mini, Cat. No. 539791)
      • ProteoExtract® Native Membrane Protein Extraction Kit (M-PEK, Cat. No. 444810).

      With the exception of the collagenase enzyme the set contains all reagents required for efficient dissociation of tissue samples. The goal of the cell isolation procedure is to maximize the yield of viable dissociated cells, so the end-user must select the collagenase appropriate for each tissue with respect to the type of tissue, the species of origin, and the age of the animal. A list of enzymes is provided below in Materials Required but Not Provided.
      Principles of the assayThe Calbiochem® ProteoExtract® Tissue Dissociation Buffer Kit is optimized to isolate viable cells from fresh tissue. Fresh tissue is first minced into small pieces, washed with PBS, and incubated with Tissue Dissociation Buffer and a collagenase of choice. Following incubation the tissue is strained using a tissue sieve. Isolated cells are ready for protein extraction.
      Materials providedThere are sufficient reagents for 10 dissociation reactions of 1 g tissue each.

      • 10X Tissue Dissociation Buffer (Kit Component No. KP31785): 1 bottle, 50 ml
      • Protease Inhibitor Cocktail (Kit Component No. KP31786): 1 vial, 1 ml
      • Tissue Dissociation Buffer Additive (Kit Component No. KP31787): 1 vial, 100 µl
      Materials Required but not provided• Collagenase: the collagenase should efficiently and specifically dissociate the tissue type of choice. Based on internal testing, a minimum of 10,000 units (~8-10 mg) collagenase is recommended per gram of tissue. The exact amount will depend on the specific enzyme and type of tissue. The list of enzymes below represents possible options used successfully with this kit. Please always refer to the manufacturer's recommendations and specifications for use.

      Table 1: Collagenases Used With Various Tissues


      • Kit for downstream processing (optional; e.g. ProteoExtract® Subcellular Proteome Extraction Kit, Cat. No. 539790, ProteoExtract® Subcellular Proteome Extraction Kit Mini, Cat. No. 539791, or ProteoExtract® Native Membrane Protein Extraction Kit, Cat. No. 444810)
      • Hemocytometer
      • Phosphate Buffered Saline (PBS)
      • Pasteur or micropipet
      • Microscope
      • Trypan blue (e.g. 0.18% in PBS)
      • 15 ml conical centrifuge tubes
      • Thermo mixer
      • Sieve
      • Scalpel or scissors
      • Refrigerated centrifuge (minimum rcf = 350 g)
      • Petri dish
      • 37°C incubator with gentle agitation
      Reagent preparation• 1X Tissue Dissociation Buffer: Prepare 50 ml 1X Tissue Dissociation Buffer by adding 5 ml 10X Tissue Dissociation Buffer to 45 ml high quality water. Add 8.5 µl Tissue Dissociation Buffer Additive and mix. • Tissue Dissociation Solution: Dissolve an appropriate amount of collagenase in 10 ml 1X Tissue Dissociation Buffer just prior to use. • 1X Tissue Dissociation Buffer with Protease Inhibitors: Add 100 µl Protease Inhibitor Cocktail to the remaining 40 ml 1X Tissue Dissociation Buffer and mix.
      Detailed protocolTissue Dissociation

      1. Cut 1 g fresh tissue into small pieces (e.g., ~10-20 pieces per g tissue) using a scalpel or scissors.
      2. Wash the minced tissue 2 times in PBS using gentle agitation in a petri dish.
      3. Transfer the minced tissue to a 15 ml conical tube.
      4. Add 10 ml Tissue Dissociation Solution and incubate for 50 min at 37°C with gentle agitation (e.g. using a hybridization oven).
      5. Following incubation strain the sample using a sieve and discard the undissociated tissue fragments.
      6. Transfer the dissociated tissue (i.e. the material that passes through the sieve) to a fresh tube and add 1X Dissociation Buffer with Protease Inhibitors to a final volume of 13 ml.
      7. Centrifuge for 5 min at 350 g and 4°C to pellet the dissociated tissue.
      8. Resuspend the pellet in 10 ml 1X Dissociation Buffer with Protease Inhibitors and centrifuge as in step 7.
      9. Resuspend the pellet in 5 ml 1X Dissociation Buffer with Protease Inhibitors.

      Viability Staining and Counting Tissue-Derived Cells

      It is important to quantitate the number of cells obtained from each dissociation step in order to effectively evaluate the results. The use of a cell counting chamber (e.g. hemocytometer) for quantitating the number of cells and trypan blue for viability is recommended.

      Figure 1: Neubauer Hemocytometer

      For calculation of cell density count 1 square millimeter and calculate as described in below.


      1. Carefully clean the counting chamber surface and the coverslip of the hemocytometer with 70% isopropanol and allow to air dry. Be careful not to scratch the surfaces.
      2. Wet the sides of the coverslip with reagent grade water and align the coverslip over the counting chamber.
      3. Mix the dissociated cell suspension and combine 20-50 µl cells with an equal volume of trypan blue and mix thoroughly. Immediately transfer the cell suspension to the counting chamber by placing the tip of the pipet at the edge of the chamber and allowing the chamber to fill completely via capillary action. Do not over- or under-fill the chamber.
      4. Repeat this procedure using a second aliquot of sample for the second chamber on the opposite side of the hemocytometer.
      5. Place the hemocytometer on the microscope stage and, using the 10X objective, focus on the counting chamber grid lines. Adjust the contrast as needed to clearly see both the grid and the dispersed cells.
      6. Adjust the field area by slowly moving the slide to obtain a central grid bounded by three lines on all sides. Count the total number of cells present in this 1 mm2 area (see figure 1) including those cells that are on the top and left borders and excluding those on the right and bottom borders.
      7. For accuracy count at least 100-500 cells. Depending upon yield and density more or fewer areas may be counted.
      8. Repeat the count for the second chamber. If no second chamber exists, the slide should be cleaned and the process repeated.
      Calculations

      Table 2: Calculating Number of Cells
      Assay characteristics and examples

      Figure 2: Western Blot Analysis of Fractionated Proteins Following Tissue Dissociation

      Tissue dissociation of rat pancreatic tissue and human pancreatic tissue was carried out according to the protocol outlined above. 1.3 x 107 dissociated cells were extracted using the ProteoExtract® Subcellular Proteome Extraction Kit protocol (Cat. No. 539790), incorporating the protocol changes as outlined above. Proteins from the cytosolic fraction, the membrane fraction, the nuclear fraction, and the cytoskeletal fraction were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to the indicated markers.
      Technical AppendixNotes on Subcellular Fractionation Using S-PEK The following deviations from the ProteoExtract® Subcellular Proteome Extraction Kit (Cat. Nos. 539790 or 539791) protocol for tissue culture cells should be applied when using dissociated cells: • As a starting point for all tissues use 1.3 x 107 to 1.8 x 107 viable dissociated cells • Follow the procedure for suspension (or non-adherent) cells • Tissues, particularly cancer tissue, consist of different cell types and frequently contain a high level of adhesion molecules. To optimize the protein yield of the cytoskeletal fraction incubate this fraction at 90°C for 5 min using a thermomixer. • The tissue dissociation generally results in more cellular debris than when starting with cultured cells, therefore an additional centrifugation step is recommended: centrifuge all fractions for 10 min at 4°C, 40,000 g. The supernatant may then be used for downstream analysis.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Biosciences, Inc.
      ProteoExtract® is a registered trademark of KGaA, Darmstadt, Germany.