Protein sample preparation

Transcription

1 GE Healthcare Life Sciences Protein sample preparation Selection Guide

2 Protein sample preparation workflows Enrichment and purification formats Mag Sepharose para-magnetic beads are designed for simple enrichment of proteins and small-scale purification or screening. Mag Sepharose beads slurry volume can be varied in a linear manner to match capacity needs, and large sample volumes of low-expressed target protein can be enriched and concentrated. Sample volume from a few microliters to about 50 ml can be used with Mag Sepharose. SpinTrap prepacked microspin columns enable fast small-scale enrichment, depletion, or purification of target protein using a bench top microcentrifuge. SpinTrap columns are prepacked with chromatography medium. Multiple plates samples can be processed simultaneously. Almost all protein samples need further preparation after collection. The quality of sample preparation is crucial to get the best possible analytical results. This selection guide helps you to find the appropriate methods and products to obtain proteins for further studies in scales from nanograms to milligrams of purified protein. Three separate workflows are available for different sources of target protein; recombinant proteins, antibodies, and proteins from natural sources. These workflows are further refined and adapted to suit different number of samples, from a few to many such as in screening applications. In sample preparation, all the targeted protein from the initial biological source must be transferred into a homogeneous solution. This can be performed either with preserved biological activity or, if required, in denatured state. The initial preparations include operations such as sample collection, stabilization, and extraction. After the initial preparation, a second step is often needed to selectively capture the target protein using enrichment or purification methods. For the study of proteins in plasma and serum, depletion of high-abundant proteins is often needed to be able to analyze low-abundant proteins. Sample preparation also includes removal of interfering nonprotein contaminants present in the initial source, amendment of noncompatible buffer compositions, and adjustment of the total protein concentration and volume. Our products address most parts of the sample preparation process for extraction, stabilization, and enrichment/purification of target molecules. A range of products are available, covering any scale and throughput of protein sample preparations: Mag Sepharose*, MultiTrap*, SpinTrap*, GraviTrap*, HiTrap* and HiPrep* are different formats for reliable enrichment, screening, and small-scale purification of any target proteins. All formats are based on the established GE Life Science s chromatography media, which offer efficient protocols resulting in high enrichment factor, purity, and recovery of your target proteins. Additional relevant products are also stated, where appropriate. Our protocols are optimized to suit typical situations. If needed, critical steps can be easily refined following the guidelines in the Instructions. MultiTrap plates are prepacked 96-well s for high-throughput processing of multiple samples; typically used for expression screening of proteins or optimization of conditions for capture and elution of target molecules. Each well is prepacked with a fixed volume of affinity chromatography medium. MultiTrap can be used manually or in automated systems, together with vacuum or centrifugation. GraviTrap columns are prepacked gravity-flow columns for purification and clean-up of proteins and other biomolecules without the need for a purification system. GraviTrap columns are packed with 1 or 2 ml of chromatography medium, allowing capture of milligram amounts of target protein. PD-10 Desalting Columns, PD MiniTrap*, and PD MidiTrap* are prepacked gravity-flow columns designed for clean-up, desalting, and buffer exchange of biomolecules, allowing sample volumes between 0.1 and 3.5 ml. HiTrap columns are prepacked 1 or 5 ml columns that can be used either with a syringe, laboratory pump, or a chromatography system. A broad range of columns is available for purification of all different kinds of proteins. Two or more columns can easily be connected in series to increase the capacity making the loading sample volume very flexible. HiPrep 16/10 columns are prepacked 20 ml columns for use with chromatographic systems. They enable binding of high milligram to gram amounts of proteins and are useful tools for preparative purifications. The columns are available for purification of many different proteins, and a broad range of sample volumes can be loaded. 2 3

3 Sample preparation for analytical purposes Sample Recombinant proteins Antibodies Natural sources Scale Scale Scale mg µg µg ng Screening mg-µg mg µg µg ng Screening mg µg µg ng Screening Tagged protein Biotinylated protein Production of antigen Tissues Cells Biological fluids 41 His GST MBP Strep II Recombinant Natural Source Disintegration Centrifugal preparations Gene construct, choice of cell line, cultivation, harvest Follow Recombinant proteins workflow Follow Natural sources workflow Cell lysis, centrifugation prep steps, buffer adjustments Collection of fluids Cell lysis, centrifugation prep steps, buffer adjustments Immunization Production antigen chromatography medium Polyclonal Monoclonal Global proteomics Proteins expression detection Collection of serum Hybridoma cell line: Preparation, cultivation and harvest Preparation of known target protein(s) Protein purification Page 6-7 Page 8-9 Page 10 Page 11 Page 12 Page 13 Page Page Page

4 Recombinant proteins Microgram to milligram scale Untagged Follow work flow for Natural sources page 14 Untagged Follow work flow for Natural sources page 15 Tagged proteins Biotinylated protein Tagged proteins Biotinylated protein Membrane protein preparation Soluble protein preparation Membrane protein preparation Soluble protein preparation Solubilization Solubilization Histidine tag GST tag MBP tag Strep-tag** II Biotin tag Histidine tag GST tag MBP tag Strep-tag** II Biotin tag Tag cleavage Tag cleavage Concentration, buffer exchange, gel filtration 1 Concentration, buffer exchange, gel filtration Column format is volume dependent 1 Column format is volume dependent 6 7

5 Recombinant proteins Nanogram to microgram scale Untagged Follow work flow for Natural sources page 16 Untagged Follow work flow for Natural sources page 17 Tagged proteins Biotinylated protein Tagged proteins Biotinylated protein Biotinylated protein Membrane protein preparation Soluble protein preparation Membrane protein preparation Soluble protein preparation Solubilization Solubilization Histidine tag GST tag MBP tag Strep-tag** II Biotin tag Histidine tag GST tag MBP tag Strep-tag** II Biotin tag Tag cleavage Tag cleavage Concentration, buffer exchange, gel filtration 1 Concentration, buffer exchange, gel filtration Column format is volume dependent 1 Column format is volume dependent 8 9

6 Recombinant proteins Screening Antibodies Microgram to milligram scale Untagged Follow work flow for Natural sources page 18 Tagged proteins Biotinylated proteins Hybridoma cell line: Preparation, cultivation and harvest Collection of serum Hybridoma cell line: Preparation, cultivation and harvest Collection of serum Membrane protein preparation Soluble protein preparation Detergent screening 42 Protein A/G purification Antigen chromatography Protein A/G purification Antigen chromatography Histidine tag GST tag MBP tag Strep-tag** II Biotin tag Tag cleavage Concentration, buffer exchange, gel filtration Concentration, buffer exchange, gel filtration Concentration, buffer exchange, gel filtration ELISA Western blotting Immunohistochemistry Immunoprecipitation ELISA Western blotting Immunohistochemistry Immunoprecipitation 1 Column and 96-well plate format is volume dependent 10 11

7 Antibodies Antibody Nanogram to microgram scale Screening Hybridoma cell line: Preparation, cultivation and harvest Collection of serum Hybridoma cell line: Preparation, cultivation and harvest Collection of serum Hybridoma cell line: Preparation, cultivation and harvest Collection of serum Protein A/G purification Antigen chromatography Protein A/G purification Antigen chromatography Protein A/G purification Antigen chromatography Concentration, buffer exchange, gel filtration Concentration, buffer exchange, gel filtration Concentration, buffer exchange, gel filtration ELISA Western blotting Immunohistochemistry Immunoprecipitation ELISA Western blotting Immunohistochemistry Immunoprecipitation ELISA Western blotting Immunohistochemistry Immunoprecipitation 12 13

8 Natural sources Microgram to milligram scale Preparation of known target protein(s) Preparation of known target protein(s) Membrane protein Other protein Phosphoprotein Membrane protein Other protein Phosphoprotein Detergent solubilization BSA/IgG depletion Phosphopeptide enrichment Detergent solubilization BSA/IgG depletion Phosphopeptide enrichment Affinity IMAC Pull-down HIC IEX Affinity IMAC Pull-down HIC IEX Polishing step Polishing step

9 Natural sources Nanogram to microgram scale Natural sources Screening Meny samples Preparation of known target protein(s) Preparation of known target protein(s) Preparation of known target protein(s) Membrane protein Other protein Phosphoprotein Membrane protein Other protein Phosphoprotein Membrane protein Other protein Phosphoprotein Detergent solubilization BSA/IgG depletion Phosphopeptide enrichment Detergent solubilization BSA/IgG depletion Phosphopeptide enrichment Detergent screening BSA/IgG depletion Phosphopeptide enrichment Affinity IMAC Pull-down HIC IEX Affinity IMAC Pull-down HIC IEX Affinity IMAC Pull-down HIC IEX Polishing step Polishing step Polishing step

10 Ordering information Product Size/quantity Code no. Product Size/quantity Code no. Product Size/quantity Code no. Product Size/quantity Code no. Product Size/quantity Code no. Product Size/quantity Code no. pgex vectors pgex-4t-1 25 µg pgex-4t-2 25 µg pgex-4t-3 25 µg pgex-5x-1 25 µg pgex-5x-2 25 µg pgex-5x-3 25 µg pgex-2tk 25 µg pgex-6p-1 25 µg pgex-6p-2 25 µg pgex-6p-3 25 µg pgex-2t 25 µg pgex-3x 25 µg pgex -1λT EcoRI/BAP 5 µg GST vector primers for sequencing pgex 5 Sequencing Primer pgex 3 Sequencing Primer E. coli BL MK13K07 Helper Phage Lysis/Protein extraction Sample Grinding Kit Yeast Protein Extraction Buffer Kit Mammalian Protein Extraction Buffer 500 ml Enzyme reguation Protease Inhibitor Mix Nuclease Mix Detection Anti-His Antibody Anti-GST Antibody 0.5 ml, 50 detections GST Detection Module GST 96-well Detection Module 5 plates Cleavage PreScission Protease Thrombin Factor Xa Buffer kits Membrane Protein Purification Kit His Buffer Kit GST Buffer Kit Protein A/G SpinTrap Buffer Kit NHS HP SpinTrap Buffer Kit Streptavidin HP SpinTrap Buffer Kit Ab Buffer Kit Protein depletion Albumin & IgG Depletion SpinTrap HiTrap Albumin & IgG Depletion 2 1 ml Desalting/Buffer exchange/cleanup Disposable PD-10 Desalting PD MidiTrap G PD MiniTrap G PD SpinTrap G PD MultiTrap G well PD MidiTrap G PD MiniTrap G HiTrap Desalting 5 5 ml HiPrep 26/10 Desalting 1 53 ml Superdex* 200 5/150 GL 1 3 ml 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 Protein concentration Vivaspin** 500 MWCO Vivaspin 500 MWCO Vivaspin 500 MWCO Vivaspin 500 MWCO Vivaspin 500 MWCO Vivaspin 500 MWCO Vivaspin 2 MWCO Vivaspin 2 MWCO Vivaspin 2 MWCO Protein concentration (cont.) Vivaspin 2 MWCO Vivaspin 6 MWCO Vivaspin 6 MWCO Vivaspin 6 MWCO Vivaspin 6 MWCO Vivaspin 6 MWCO Vivaspin 6 MWCO Vivaspin 20 MWCO Vivaspin 20 MWCO Vivaspin 20 MWCO Vivaspin 20 MWCO Vivaspin 20 MWCO Vivaspin 20 MWCO Antibody purification/pull-down HiTrap Protein A HP 5 1 ml HiTrap Protein A HP 5 5 ml HiTrap rprotein A FF 5 1 ml HiTrap rprotein A FF 5 5 ml MAbTrap Kit 1 1 ml rprotein A GraviTrap 10 1 ml Protein A Mag Sepharose µl Protein A Mag Sepharose µl Protein A HP SpinTrap Protein A HP MultiTrap 4 96 well Protein A Mag Sepharose Xtra 2 1 ml Protein A Mag Sepharose Xtra 5 1 ml Ab SpinTrap Protein G GraviTrap 10 1 ml HiTrap Protein G HP 5 1 ml HiTrap Protein G HP 5 5 ml Protein G Mag Sepharose 500 µl Protein G Mag Sepharose µl Protein G HP SpinTrap Protein G HP MultiTrap 4 96 well Protein G Mag Sepharose Xtra 2 1 ml Protein G Mag Sepharose Xtra 5 1 ml rprotein A/Protein G GraviTrap 10 1 ml HiTrap NHS-activated HP 5 1 ml Antibody purification/pull-down (cont.) NHS Mag Sepharose µl NHS HP SpinTrap Streptavidin Mag Sepharose 2 1 ml Streptavidin Mag Sepharose 5 1 ml HiTrap Streptavidin HP 5 1 ml Streptavidin HP SpinTrap Streptavidin HP MultiTrap 4 96 well Other affinity chromatography methods HiTrap Blue HP 5 1 ml HiTrap Blue HP 1 5 ml HiTrap Heparin HP 5 1 ml HiTrap Heparin HP 5 5 ml HiPrep Heparin FF 16/ ml HiTrap Con A 4B 5 1 ml HiTrap Con A 4B 5 5 ml TiO 2 Mag Sepharose 500 µl TiO 2 Mag Sepharose µl Phos SpinTrap Fe IEX HiTrap SP HP 5 1 ml HiTrap SP HP 5 5 ml HiTrap Q HP 5 1 ml HiTrap Q HP 5 5 ml HiTrap Q FF 5 1 ml HiTrap Q FF 5 5 ml HiTrap SP FF 5 1 ml HiTrap SP FF 5 5 ml HiTrap DEAE FF 5 1 ml HiTrap DEAE FF 5 5 ml HiTrap CM FF 5 1 ml HiTrap CM FF 5 5 ml HIC HiTrap Phenyl FF (high sub) 5 1 ml HiTrap Phenyl FF (high sub) 5 5 ml HiTrap Phenyl FF (low sub) 5 1 ml HiTrap Phenyl FF (low sub) 5 5 ml HiTrap Phenyl HP 5 1 ml HIC (cont.) HiTrap Octyl FF 5 5 ml HiTrap Butyl FF 5 1 ml HiTrap Butyl FF 5 5 ml HiTrap Butyl-S FF 5 1 ml HiTrap Butyl-S FF 5 5 ml HiTrap Butyl HP 5 1 ml HiTrap Butyl HP 5 5 ml Selection Kits HiTrap HIC Selection Kit 7 1 ml HiTrap IEX Selection Kit 7 1 ml HiTrap IEX/Capto* Selection Kit 5 1 ml GST-tagged protein capture GSTrap* HP 5 1 ml GSTrap HP 5 5 ml GSTrap FF 5 1 ml GSTrap FF 5 5 ml GSTrap 4B 5 1 ml GSTRap 4B 5 5 ml GSTPrep* FF 16/ ml GST SpinTrap GST MultiTrap FF 4 96 well GST MultiTrap 4B 4 96 well GST GraviTrap GST Bulk Kit Histidine-tagged protein capture HisTrap* FF crude 5 1 ml HisTrap FF crude 5 5 ml HisTrap FF crude Kit 1 kit HisTrap HP 5 1 ml HisTrap HP 5 5 ml HisTrap FF 5 1 ml HisTrap FF 5 5 ml HisPrep* FF 16/ His GraviTrap His GraviTrap Kit His Mag Sepharose Ni 2 1 ml Histidine-tagged protein capture (cont.) His SpinTrap His SpinTrap Kit His MultiTrap FF 4 96 well His MultiTrap HP 4 96 well Strep-tag II protein purification StrepTrap* HP 5 1 ml StrepTrap HP 5 5 ml MBP-tagged protein purification MBPTrap* HP 5 1 ml MBPTrap HP 5 5 ml PreDictor* plates PreDictor AIEX Screening Plate 20 µl, well PreDictor CIEX Screening Plate 20µl, 4 96 well Accessories MagRack MagRack Maxi Collection Plate 500 µl V-bottom MiniSpin Adapter MidiSpin Adapter PD-10 Spin Adapter HiTrap Benzamidine FF (high sub) 2 1 ml Vivaspin 2 MWCO HiTrap NHS-activated HP 1 5 ml HiTrap Phenyl HP 5 5 ml His Mag Sepharose Ni 5 1 ml HiTrap Benzamidine FF (high sub) 5 1 ml Vivaspin 2 MWCO NHS Mag Sepharose 500 µl HiTrap Octyl FF 5 1 ml His Mag Sepharose Ni 10 1 ml

11 About the selection guides Product index Three different work flows, for different sources of target proteins, are available 1 His Mag Sepharose Ni 42 Membrane Protein Purification Kit Recombinant proteins Antibodies Proteins from natural sources 2 Streptavidin Mag Sepharose 3 NHS Mag Sepharose 4 Protein A Mag Sepharose 43 Anti-His Ab 44 Anti-GST Ab 45 GST Detection Module Each workflow contains three separate schemes; purifications in microgram to milligram scale, nanogram to microgram scale, or for screening purposes. 5 Protein A Mag Sepharose Xtra 6 Protein G Mag Sepharose 7 Protein G Mag Sepharose Xtra 46 His Buffer Kit 47 GST Buffer Kit 48 PreScission Proetase At the bottom of each track, reference is given to spreads with more detailed workflows for each source and scale. All of these spreads contains two workflows, one for few, and one for many samples. In these workflows, suitable products are indicated with numbers. A list of all products corresponding to the numbers can be found on the right, Ordering information for products is found at the end of the Selection guide. To use this Selection guide, start on pp 4-5, where the three workflows are found. 8 TiO 2 Mag Sepharose 9 His SpinTrap 10 GST SpinTrap 11 His SpinTrap Kit 12 PD SpinTrap G Protein A HP SpinTrap 14 Protein G HP SpinTrap 15 NHS HP SpinTrap 16 Albumin & IgG Depletion SpinTrap 17 Phos SpinTrap Fe 18 Streptavidin HP SpinTrap 19 His MultiTrap 20 GST MultiTrap 21 Streptavidin HP MultiTrap 22 PD MultiTrap G Protein A HP MultiTrap 24 Protein G HP MultiTrap 25 His GraviTrap 26 GST GraviTrap 27 PD-10 Desalting Columns 28 PD MidiTrap 29 PD MiniTrap 49 Thrombin 50 Factor Xa 51 Vivaspin 52 Mini Dialysis Kit 53 MagRack 6 54 MagRack Maxi 55 HisTrap 56 GSTrap 57 MBPTrap HP 58 StrepTrap HP 59 HiTrap Streptavidin HP 60 HiTrap Albumin & IgG Depletion 61 HiTrap Blue HP 62 HiTrap Heparin HP 63 HiPrep Heparin FF 16/10 64 HiTrap Benzamidine FF (high sub) 65 HiTrap Con A HP 66 HiTrap Butyl 67 HiTrap Desalting 68 HiTrap Phenyl 69 HiTrap Protein A 70 HiTrap Protein G GE Life Science s sample preparation products 30 rprotein A GraviTrap 71 HiTrap NHS-activated HP 31 Protein G GraviTrap 72 HiTrap Octyl 32 rprotein A/Protein G GraviTrap 73 HiTrap Q 33 E. coli B HiTrap SP 34 pgex vector 75 HiTrap DEAE FF 35 MK13K07 Helper Phage 76 HiTrap CM FF 36 GST vector primer for sequencing 77 HiTrap HIC Selection Kit 37 Yeast Protein Extraction Buffer Kit 78 HiTrap IEX Selection Kit 38 Mammalian Protein Extraction Buffer 79 HisPrep FF 16/10 39 Protease Inhibitor Mix 80 GSTPrep FF 16/10 40 Nuclease Mix 81 HiPrep 26/10 Desalting 41 Sample Grinding Kit

12 For contact information for your local office, please visit, GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE, imagination at work and GE monogram are trademarks of General Electric Company. * Capto, GraviTrap, GSTPrep, GSTrap, HiPrep, HisPrep, HisTrap, HiTrap, MAbTrap, MBPTrap, MidiTrap, MiniTrap, MultiTrap, PreDictor, PreScission, Sepharose, SpinTrap, StrepTrap, Superdex, and Vivaspin are trademarks of GE Healthcare companies. ** All third-party trademarks are the property of their respective owners. pgex Vectors: pgex Vectors are to be used for scientific investigation and research and for no other purpose whatsoever and a license for commercial use of the licensed products and the processes claimed in US patent 5,654,176 and equivalent patents and patent applications in other countries must be negotiated directly with Millipore Corp (formerly Chemicon International Inc) by the purchaser prior to such use. US patent numbers 5,284,933 and 5,310,663, and equivalent patents and patent applications in other countries (assignee: Hoffman La Roche, Inc) relate to the purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues (commonly known as the histidine-tag technology). Any customer that wishes to use Chelating Sepharose Fast Flow, Ni Sepharose 6 Fast Flow or IMAC Sepharose 6 Fast Flow for non-research/commercial applications under these patents is requested to contact Hoffman-La Roche AG, Corporate licensing, attention Dr Andreas Maurer, CH-4070 Basel, Switzerland, telephone , fax , for the purpose of obtaining a license. StrepTrap HP and StrepTactin Sepharose High Performance are covered by US and equivalent patents and patent applications in other countries. The purchase of StrepTrap HP and StrepTactin Sepharose High Performance includes a license under such patents for non-profit and in-house research only. Please contact IBA (info@iba-go.com) for further information on licenses for commercial use of StrepTactin General Electric Company All rights reserved. First published June 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 local GE Healthcare representative for the most current information. GE Healthcare Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ , USA GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg, Germany GE Healthcare Bio-Sciences KK Sanken Bldg., , Hyakunincho, Shinjuku-ku, Tokyo, Japan AA 06/2011