2-D Fractionation Kit

Transcription

1 GE Healthcare 2-D Fractionation Kit Ettan sample preparation kits and reagents. Product Booklet Code:

2 Page finder 1. Legal 3 2. Handling Safety warnings and precautions Storage Expiry 4 3. Components 5 4. Overview 6 5. Critical parameters 8 6. Related products referred to in protocol D Fractionation Kit Protcol Before use Preparation of the Soluble Protein Component and the Insoluble Protein Fraction Processing the Soluble Protein Component Processing the Soluble Protein Component Fractions (Pellets I to VI) for 2-D electrophoresis Processing the Insoluble Protein Fraction for 2-D electrophoresis Optional modifications to the Protocol Quality control Related products 19 2

3 1. Legal GE, imagination at work and GE monogram are trademarks of General Electric Company. Ettan is a trademark of GE Healthcare companies. For details on Ettan range of sample preparation products and Ettan DIGE products*, see or contact your local GE Healthcare office. *Ettan DIGE is a unique system that brings a unique level of statistical confidence and reliability to 2-D electrophoresis results. This product is manufactured by G-Biosciences, for worldwide distribution by GE Healthcare General Electric Company All rights reserved. First published 2007 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your GE Healthcare representative for the most current information. http// GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK 3

4 2. Handling 2.1. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice Storage For convenience, the kit can be stored at ambient on receipt. However, some components need to be chilled before first use and/or stored at lower temperatures after first use. See instructions specified, in the components list on page Expiry The components of these products are stable for at least 12 months when stored under the recommended conditions. 4

5 3. Components Lysis Buffer: Once opened, store at 2 8 C. Fraction Precipitant: Store at ambient. Precipitant: Store at ambient. Co-Precipitant: Store at ambient. Wash Buffer: Place at -20 C for 1 2 hours before first use and store at that temperature thereafter. Solubilizer: Store powder at ambient. Diluent: Once opened store at 2 8 C. 5

6 4. Overview A major challenge in protein discovery and the analysis of the proteome is the preparation of samples for 2-D gel analysis. In whole cell lysates, proteins exist in a wide dynamic range of concentrations. Consequently, abundant proteins mask identification of less abundant proteins. An effective proteome analysis will require separation of abundant proteins and the enrichment of less abundant ones to bring the latter into a detectable range. Fractionation has the following advantages: It reduces the number of protein species present in each fraction. This increases the mass of each protein that can be loaded on to a gel so bringing the low abundance proteins into the dynamic range of detection. It produces less crowded individual protein maps, simplifying analysis and interpretation. The 2-D Fractionation kit exploits the property of protein molecules to precipitate in response to the changes in solvent composition (i.e., ionic strength, ph, temperature). The solvent composition is changed in a step-wise fashion such that a series of protein fractions is produced. The kit has been optimized around a protocol that produces a set of 7 fractions (see Figure 1). However, there is the option to optimize these to your own needs. This kit is suitable for 10 separate preparations each of 50 mg of wet cells. 6

7 Tissue / Cells Lysis buffer Sonicate Spin Supernatant Pellet 0.1 ml Fraction Precipitant, incubate, spin Pellet = Fraction I Supernatant Re-wash Insoluble fraction 0.1 ml Fraction Precipitant, incubate, spin Solubilize 2D gel Pellet = Fraction II Supernatant 0.2 ml Fraction Precipitant, incubate, spin Pellet = Fraction III Supernatant Further additions of Fraction Precipitant or Precipitant: Pellets = Fractions IV, V, VI (Wash VI), dialyse all in buffer of choice 2D gels Figure 1: Schematic representation of the Ettan 2-D Fractionation kit 7

8 5. Critical parameters Read the entire protocol thoroughly before using the kit. Once the cell or tissue sample has been lysed and the supernatant (Soluble Protein Component) has been collected, fractionation of the proteins within the solution should be completed within 2 3 hours. Storage and/or freezing of the soluble protein component may lead to precipitation of some soluble proteins. For reproducibility between different extractions of the same tissue or cell type, it is important to use the same mass of starting material each time. 8

9 6. Related products referred to in the protocol Protease Inhibitor Mix (code ) Nuclease Mix (code ) Sample grinding kit (code ) Mini Dialysis Kits (codes , , and ) 9

10 7. 2-D Fractionation Kit Protocol 7.1. Before use 1. Transfer the Wash Buffer to -20 C for 1 2 hours before use. Note: It is important to prepare these solutions ready for use and fractionate the soluble proteins within 2 3 hours. Storage and freezing of the soluble proteins may lead to precipitation of some proteins and hence loss of reproducibility i.e. such proteins will end up distributed differently across the fractions. 2. Place Lysis Buffer in a wet-ice bath to keep chilled. Note: If the inhibition of protease activity is required, add a cocktail of protease inhibitors (e.g Protease Inhibitor Mix; see Related Products) into the Lysis Buffer. Likewise, if the removal of nucleic acids is required, add an appropriate amount of DNase and RNase e.g. Nuclease Mix (see Related Products) into the Lysis Buffer. 3. Make sure that there is no crystal formation in the Fraction Precipitant reagent. Gentle warming of the bottle with the hands should help dissolve any crystals that are present. 4. Prepare the Solubilizer Buffer as follows: Note: Use only freshly made Solubilizer Buffer; discard any that is unused. a) Shake the bottle of Solubilizer powder for seconds. b) Weigh an appropriate amount (each gram of dry reagent will produce 2 ml buffer). c) For each 1 gram of dry reagent add 1.1 ml of Diluent. d) Mix well by vortexing periodically to obtain a clear solution. 10

11 7.2. Preparation of the Soluble Protein Component and the Insoluble Protein Fraction 1. Sonicate the cells or tissue in Lysis Buffer at 0 4 C (e.g. in a wet ice bath). Use 0.5 ml Lysis Buffer for 100 mg tissue or 0.05 g of wet cells. For plants, use 2 ml of lysis buffer per 1 gram of tissue. Sonication should be performed in multiple short bursts to prevent heating of the sample. Notes: The sample to buffer volume ratio can be adjusted depending on the scale of the preparation. However, for reproducibility, use the same mass of cells/tissue each time a particular fractionation is repeated. Bacterial cells generally require longer sonication than animal cells and tissues. Yeast cells require even more vigorous sonication; addition of glass beads in the yeast cell suspension greatly facilitates disruption of yeast cells. Soft tissues may be homogenized using a pestle-tube homogenizer, electrical blender or a grinder. The Sample Grinding Kit (see Related Products) may be used for efficient grinding of tissues. Like sonication, care must be taken to prevent heating during homogenization. 2. Centrifuge the homogenate at x g for 30 minutes at 4 C to pellet the cellular debris. 3. Using a pipette, transfer the clear supernatant to a clean 2 ml tube without disturbing the pellet. 4. Suspend the pellet in 1/4 the volume of Lysis Buffer used in step 1, sonicate the pellet once briefly (30 seconds). Repeat steps 2 and 3, collect the clear supernatant and pool with the first supernatant. 5. Label the pooled supernatant as Soluble Protein Component and store on ice. 11

12 Note: Do not freeze the Soluble Protein Component at this stage as it may precipitate some proteins, affecting the subsequent fractionation process. 6. Suspend the pellet in 0.5 ml Lysis Buffer and vortex for 60 seconds. Centrifuge at x g for 15 minutes at 4 C. Remove and discard the wash. 7. Label the washed pellet as Insoluble Protein Fraction and store on ice Processing the Soluble Protein Component The Soluble Protein Component can now be separated in to several fractions by the sequential addition of precipitation reagents. It is suggested that the protocol is followed as written in the first instance. Depending on the particular objective, it is possible, after analysis of the samples by gel electrophoresis, to adjust the volumes of precipitant added at each stage to either further separate certain proteins from others or to concentrate a given protein into a single fraction. A. Fraction I i) Place 0.5 ml of the Soluble Protein Component into a 2 ml tube and add 0.1 ml of Fraction Precipitant reagent drop-wise to it. Close the tube, invert it a few times and incubate it on ice for 5 minutes. Note: If you wish to use a larger or smaller volume of Soluble Protein Component, the volumes of Fraction Precipitant reagent may be adjusted accordingly. ii) Centrifuge at x g, 4 C for 10 minutes. Transfer the supernatant to a clean 2 ml tube. iii) Centrifuge the pellet again for 5 10 seconds at x g and combine any residual supernatant with that from step ii). Label the pellet as Fraction I and store on ice. 12

13 B. Fraction II i) Add a further 0.1 ml of Fraction Precipitant reagent. Close, invert and incubate on ice as for 5 minutes. ii) Centrifuge at x g, 4 C for 10 minutes. Transfer the supernatant to a clean 2 ml tube. iii) Centrifuge the pellet again for 5 10 seconds at x g and combine any residual supernatant with that from step ii). Label the pellet as Fraction II and store on ice. C. Fraction III i) Add a further 0.2 ml of Fraction Precipitant reagent. Close, invert and incubate on ice for 5 minutes. ii) Centrifuge at x g, 4 C for 10 minutes. Transfer the supernatant to a clean 2 ml tube. iii) Centrifuge the pellet again for 5 10 seconds at x g and combine any residual supernatant with that from step ii). Label the pellet as Fraction III and store on ice. D. Fraction IV i) Add a further 0.3 ml of Fraction Precipitant reagent. Seal, invert and incubate on ice for 5 minutes. ii) Centrifuge at x g, 4 C for 10 minutes. Transfer the supernatant to a clean 2 ml tube. iii) Centrifuge the pellet again for 5 10 seconds at x g and combine any residual supernatant with that from step ii). Label the pellet as Fraction IV and store on ice. E. Fraction V i) Add a further 0.8 ml of Fraction Precipitant reagent. Close, invert and incubate for 5 minutes. ii) Centrifuge at x g, 4 C for 10 minutes. Transfer the supernatant to a clean 15 ml tube. 13

14 iii) Centrifuge the pellet again for 5 10 seconds at x g and combine any residual supernatant with that from step ii). Label the pellet as Fraction V and store on ice. F. Fraction VI i) Add 6 ml Precipitant (Note: not Fraction Precipitant) to the supernatant from Fraction V. Seal, invert the tube a few times and incubate on ice for 10 minutes. ii) Add 3 ml Co-Precipitant. Vortex and incubate for 1 minute. Centrifuge at x g for 10 minutes at 4 C. Discard the supernatant. Centrifuge the tube again for a brief 5 10 seconds. Remove and discard any residual supernatant. iii) Add 0.2 ml Co-precipitant on top of the pellet (be careful not to disturb the pellet). Centrifuge at x g for 10 minutes at 4 C. Remove and discard the clear supernatant - be careful not to disturb the pellet. iv) Add 0.1 ml pure (e.g. sterile, non-pyrogenic) water on top of the pellet and vortex the tube for 30 seconds. v) Add 5 ml pre-chilled (-20 C) Wash Buffer and vortex the tube 4 5 times, 30 seconds each. vi) Incubate at -20 C for 30 minutes, periodically vortexing the tube to break up the pellet. Note: the pellet will not dissolve in the Wash Buffer. vii) Centrifuge at x g for 10 minutes at 4 C. viii) Discard the supernatant and invert the tube on a clean paper towel to remove residual Wash Buffer. Allow the tube to air dry. Mark the tube as Fraction VI. Note: A break can be made in the protocol at this time if needed. In this case, the pellets from the Soluble and Insoluble Fractions should be stored frozen (-15 to -30 C) until required. 14

15 7.4. Processing the Soluble Protein Component Fractions (Pellets I to VI) for 2-D electrophoresis 1. Suspend each of the pellets from the Soluble Component (Fractions I to VI) in 0.1 ml Solubilizer Buffer and vortex the suspensions 5 6 times, 30 seconds each. 2. In order to prepare Fractions I to VI for 2-D electrophoresis, samples should be dialysed against 2 3 ml of a buffer of your choice for 5-6 hours with at least one change of buffer. Notes: Mini-dialysis kits are ideal for this stage (see Related Products). We recommend the IEF Buffer below. It will allow salts to be removed when samples are dialysed against it and will render the samples suitable for iso-electric focusing (IEF). (This buffer also renders the samples compatible with Ettan DIGE labeling, allowing pre-labeling to be carried out prior to loading on to the gel if desired. See Related Products). IEF Buffer Tris 30 mm Thiourea 2 M Urea 7 M CHAPS 4% (w/v) HCl to ph Determine protein concentration of each fraction (e.g. by use of the 2-D Quant kit; see Related Products) and make an appropriate dilution in IEF Buffer or equivalent before running IEF/2-D gels. Note: The ideal loading of each sample will vary according to gel size and detection method. Typical loadings are in the range of μg per gel. 15

16 7.5. Processing the Insoluble Protein Fraction for 2-D electrophoresis 1. Suspend the Insoluble Protein Fraction pellet in 0.4 ml Solubilizer Buffer. Vortex the suspension 4 5 times, 60 seconds each, to dissolve the protein. Centrifuge at x g, 15 C for 15 minutes and collect the clear supernatant. 2. Re-suspend any residual pellet in 0.1 ml Solubilizer Buffer. Centrifuge at x g, 15 C for 15 minutes and pool the supernatant with that from step 1. Notes: Depending on the source of proteins, the volume of buffer added to some fractions may need to be increased to completely dissolve the protein but beware of diluting the protein too much for subsequent analysis (e.g. 2-D electrophoresis) that you intend to carry out. 3. Determine the protein concentration of the protein solution and make an appropriate dilution in Solubilizer Buffer or IEF buffer (see previous section) before running IEF/2-D gels Optional modifications to the protocol The stages of the protocol using the Fraction Precipitant reagent as described above fractionate the soluble protein solutions into a set of 5 fractions. As some proteins have broad precipitation ranges, those proteins may appear in more than one fraction. As stated earlier, the volumes of Fraction Precipitant reagent added at each stage of the procedure can be modified, depending on your particular interests, to enrich a given fraction with a specific protein; a 10 15% change in volume of precipitant added will significantly change the fractionation pattern achieved. Such adjustments should be done empirically following analysis of the fractionation achieved using the standard protocol. Once optimized, the volumes added in subsequent 16

17 repeat experiments should be reproduced accurately to ensure consistency of results. 17

18 8. Quality control To ensure minimal batch to batch variability, every batch of the 2-D Fractionation kit is functionally tested for the ability to reproducibly fractionate samples from mouse liver. Such fractions are then run on SDS PAGE and subjected to Western blotting to probe for the presence and distribution of tubulin. 18

19 9. Related products Sample Grinding Kit D Quant Kit Mini Dialysis Kit 1 kda cut-off, 250 μl Mini Dialysis Kit 1 kda cut-off, 2 ml Mini Dialysis Kit 8 kda cut-off, 250 μl Mini Dialysis Kit 8 kda cut-off, 2 ml D Clean-up Kit SDS Clean-up Kit Protease Inhibitor Mix Nuclease Mix Equilibration Buffer (for preparing samples for the second dimension of 2D electrophoresis) RPN

20 GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan 30, Uppsala, Sweden GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg, Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ , USA GE Healthcare Bio-Sciences KK Sanken Bldg , Hyakunincho, Shinjuku-ku, Tokyo , Japan For contact information for your local office, please visit : GE Healthcare UK Limited Amersham Place Little Chalfont, Buckinghamshire, HP7 9NA, UK imagination at work PL Rev C 06/2008