Stabilizor® 2D-GE extraction kit (TISSUE)
Contents
1. a new tube without disturbing the pellet 11 The sample is now ready for protein quantification see below and 2D GE 7 4 Protein quantification Measure the concentration of protein using a urea compatible kit e g 2 D Quant kit GE Healthcare or dilute the sample x7 to reach urea concentrations under 1M to use standard protein assays e g BCA Protein Assay kit Pierce 7 5 Downstream analysis Subsequent downstream steps are not affected by heat stabilization and conventional 2D GE protocols can be followed without alterations User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page 7 9 8 References Goodwin R J A M Lang et al 2010 Stopping the clock on proteomic degradation by heat treatment at the point of tissue excision Proteomics 10 9 1751 61 Grassl J J A Westbrook et al 2009 Preserving the yeast proteome from sample degradation Proteomics 9 20 4616 26 Robinson P K Toney et al 1995 Mass spectrometric and biological characterization of guinea pig corticotrophin Regul Pept 56 1 89 97 Rountree C B C A Van Kirk et al 2010 Clinical application for the preservation of phospho proteins through in situ tissue stabilization Proteome Sci 8 61 Scholz B K Sk ld et al 2011 Impact of temperature dependent sampling procedures in proteomics and peptidomics a characterization of the liver and pancreas post mortem degradome Mol Cel2. arning Warning Harmful if swallowed Causes skin irritation Causes serious eye irritation May cause respiratory irritation Safety Measures Avoid breathing dust IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing Note After dissolution of the Dry component into Diluent the mixture is not classified as hazardous User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page5 9 7 Specific protocol In the following step by step protocol important passages are in bold font 7 4 Heat stabilization For more information on the use of the Stabilizor system please consult the Stabilizor User Manual 1 Collect sample and place in Maintainor Tissue card 2 Heat stabilize immediately following the Stabilizor User Manual instructions If preferred samples can be frozen and heat stabilized at a later stage If necessary sub dissect samples after heat stabilization but before freezing If samples will be analyzed at a later date store samples at 80 C 7 2 Preparation of Protein Extraction Buffer Each kit contains reagents necessary to prepare 45 ml of Protein Extraction Buffer This buffer can also be used to rehydrate IPG strips for 2D GE or to extract proteins for 1D GE Protein Extraction Buffer is prepared by dissolving the Dry component with the Protein Extraction Buffer Diluent DTT is added to the Protein Ext
3. denalor Stabilizor 2D GE extraction kit TISSUE User Manual Use with DKT 0002 User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page 1 9 1 Product Description The Stabilizor 2D GE extraction kit Tissue is designed to ensure efficient homogenization and protein solubilization of heat stabilized tissues When performing 2D GE experiments with heat stabilized samples three aspects should be especially considered e Thorough homogenization e Selection and preparation of the Protein Extraction Buffer components with or without DTT following the 2D GE protocol supplied e Correct buffer to sample ratio to ensure solubilization 2 Intended use The Stabilizor 2D GE Extraction kit contains all necessary items needed for sample collection and protein extraction prior to 2D GE The Protein Extraction Buffer is supplied as three components in order to avoid carbamylation of urea and to prevent oxidation of dithiothreitol DTT The lack of enzymatic activity in heat stabilized samples ensures that samples are not at risk of enzymatic degradation during thawing or sample handling However unnecessary freeze thaw cycles and variations in sample handling should be avoided since non enzymatic processes may still affect the sample The Stabilizor 2D GE extraction kit is intended for RESEARCH USE ONLY User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page 2 9 Materials sup
4. er use NOTE Protein Extraction Buffer including DTT or not is compatible with DeStreak Rehydration Reagent GE Healthcare NOTE Carrier ampholytes or bromophenol blue dye may be added if necessary User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page 6 9 7 3 Protein extraction For easier handling perform step 1 4 on dry ice if working with frozen samples 1 Pre weigh the supplied homogenization tubes 2 Place samples in the homogenization tubes Avoid collecting samples larger than 50 mg per tube because of the risk of sample loss during open tube homogenization 3 Weigh the homogenization tubes and calculate sample weight Note Use 10 pl of Protein Extraction Buffer per milligram of sample For samples smaller than 10 mg use 100 l of Protein Extraction Buffer Crush the sample coarsely using the supplied pestle 5 Add approximately 30 of the required volume of Protein Extraction Buffer and homogenize for at least 1 minute Add remaining buffer and continue the homogenization 6 Homogenize for at least 2 minutes using the pestle continue even when no traces of coarse material remain 7 Additional micro tip sonication will improve the protein extraction Important to keep sample temperature below 37C Mix tube or vortex sample briefly before centrifuge Centrifuge the sample at top speed at least 12 000 x g for 25 min to sediment cellular debris 10 Carefully transfer supernatant into
5. it component is available online in PDF format from the Service and Support section at www denator com A S D S is only issued for the individual kit components since the final buffer is not classified as hazardous 6 1 Protein Extraction Buffer with or without DTT After preparation of buffer with or without DTT component the concentrations of hazardous compounds in the Protein Extraction Buffer are below those set by REACH regulations 1907 2006 as requiring aS D S Please consult the S D S for each individual component 6 2 Protein Extraction Buffer Diluent Concentrations of hazardous compounds in the Protein Extraction Buffer Diluent are below the limits for classification of the product as hazardous as set by REACH regulations 1907 2006 6 3 Protein Extraction Buffer Dry component Harmful Dangerous for the environment Warning Possible risk of harm to the unborn child Limited evidence of a carcinogenic effect Harmful if swallowed Toxic to aquatic organisms may cause long term adverse effects in the aquatic environment Safety Measures Wear suitable protective clothing and gloves Avoid release to the environment Refer to special instructions Safety data sheets Note After dissolution of the Dry component into Diluent the mixture is not classified as hazardous User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page 4 9 6 4 Protein Extraction Buffer DTT component W
6. l Proteomics 10 3 Mg00229MCP200 g Technical assistance For questions about this kit the use of the Stabilizor system and working with heat stabilized tissue visit www denator com for latest application details and technical support User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page 8 9 10 Contact Denator AB Arvid Wallgrens Backe 20 SE 413 46 Goteborg Sweden Phone 46 0 31 412 890 Fax 46 0 31 412 840 Mail inform denator com www denator com 2011 Denator AB Stabilizor and Maintainor are trademarks of Denator AB All third party trademarks are the property of their respective owners denalor STABILIZING THE PROTEOME User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKTO002 Page9 9
7. plied 12 x Maintainor Tissue 1x Protein Extraction Buffer Dry Component 26 g 1x Protein Extraction Buffer Diluent 25ml 3 x Protein Extraction Buffer DTT Components 23 mg 36 x Pestles for homogenization 36 x Tubes for homogenization Quick guide Stabilizor 2D GE extraction kit Tissue Required not provided User Manual Stabilizor 2D GE extraction kit Tissue available online in PDF format from the Service and Support section at www denator com Centrifuge capable of spinning 1 5ml tubes at 12 000 x g or more Sample storage tubes recommended Protein LoBind Tube Eppendorf Protein quantification kit Aliquot tubes for Protein Extraction Buffer gt 15 ml Dry ice Storage amp Disposal Store kit components prior to use in 4 C to 8 C Store prepared Protein Extraction Buffer below 18 C for up to 3 months We recommend storing this buffer in 15 ml aliquots Protein Extraction Buffer containing DTT CANNOT be stored Use Protein Extraction Buffer within 12 month from manufacturing date Follow local regulations when disposing used or unused materials User Manual 2D GE extraction kit 70014 rev 1 2011 To be used with DKT0002 Page 3 9 6 Safety Information When working with chemicals always work responsibly Wear suitable protective clothing disposable gloves and protective goggles For more information please consult the appropriate Safety Data Sheets S D S A S D S for each k
8. raction Buffer prior to use DTT reduces disulfide bonds at cysteine residues to completely denature proteins prior to 2D GE 1 Mix Dry Buffer Component and Diluent 25ml in the Dry Buffer Component bottle 2 Stir constantly until a clear solution is seen Dissolution of urea is an endothermic reaction and takes about 3 h in room temperature If faster re solubilization is required transfer the solution to a beaker standing in warm water max 37 C and stir to speed up the procedure 3 Store Protein Extraction Buffer below 18 C in aliquots 15 ml for up to 3 months If not used directly Preparing Protein Extraction Buffer with DTT Note If proteins are to be labeled with CyDye Fluor dyes for 2D DIGE analysis omit DTT from the Protein Extraction Buffer and add to the sample extract after labeling as specified by the dye manufacturer 1 Add DTT component to the Protein Extraction Buffer immediately prior to use Use one DTT component vial with 15 ml of Protein Extraction Buffer Reconstitute the DTT component by adding 1 ml of the 15 ml Protein Extraction Buffer to the DTT vial 2 Aspirate several times 3 Transfer the Protein Extraction Buffer containing the DTT component back to the Protein Extraction Buffer bottle If the entire Protein Extraction Buffer 45 ml is to be used at the same time all three 3 DTT component vials should be included in the buffer Protein Extraction Buffer containing DTT cannot be saved for lat
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