OMX-S Device for In-Gel Digestion

Transcription

1 INSTRUCTION MANUAL OMX-S Device for In-Gel Digestion (Cat. No , ) SERVA Electrophoresis GmbH Carl-Benz-Str. 7 D Heidelberg Phone , Fax info@serva.de

2 Contents 1. OMX-S device for rapid in-gel digestion General information Technical data and scope of supply Storage conditions and safety precautions 3 2. Descripiton of the OMX-S device 3 3. Detailed Protocol for the picking and tryptic in-gel 4 digestion of protein spots 3.1. Required chemicals and laboratory apparatus 4 (not supplied with the kit) 3.2. Procedure 4 4. Troubleshooting 7 5. Literature 7 1

3 1. OMX-S device for rapid in-gel digestion 1.1 General information With OMX-S, existing protocols for the in-gel digestion of protein bands can be accomplished in one single device. Sample preparation is significantly simplified and the risk of contamination is minimized. The OMX technology is cost effective, provides higher peptide yields and better quality mass spectra. The OMX-S device is a three-in-one tool featuring a combination of a Picker (P) for cutting protein bands or spots from an electrophoresis gel a Reactor (R) enabling the in-gel digestion of the protein spots a Sampler (S) for collecting the peptide solution after the digestion process. This guide features a detailed step-by-step protocol for the tryptic in-gel digestion of proteins from electrophoresis gels enabling a rapid and reproducible sample preparation Technical data and scope of supply OMX-S length: 50 mm Rotor cavity diameter required: 11 mm Maximum Reactor volume: 40 µl Maximum spin speed: x g Diameter of Picker: 1.8 mm Materials: Polypropylene and glass Resistance to chemicals: Resistant to: Not resistant to: Packaging size: contains 12 OMX-S devices and manual contains 2 OMX-S devices and manual Water, diluted acids, short chain alcohols and acetonitrile. Hydrocarbons, arenes and Halogenated hydrocarbons 2

4 1.3. Storage conditions and safety precautions OMX-S should be stored dry and clean at room temperature and protected from UVlight. OMX-S is stable for at least 1 year when stored as described. OMX-S is developed for laboratory use only. No hazardous components are contained within OMX-S. Nevertheless, appropriate safety apparel such as lab coat, eye protection and especially cleaned gloves should be worn. These precautions help to avoid sample contamination especially with traces of keratin. The OMX-S device is manufactured for single use. Until use, OMX-S should remain stored in the original package under clean conditions at room temperature in order to avoid any contamination. The device should be used within 12 months from the date of purchase. The reactor of OMX-S contains flexible components. We therefore guarantee proper efficiency only if the reactor remains closed. 2. Description of the OMX-S device Reactor (R) enables the in-gel digestion of proteins Picker (P) cuts protein bands or spots from electrophoresis gel Sampler (S) collects the peptide solution after the digestion process The OMX-S device is delivered with the Sampler (S) attached on the Picker-bearing side of the Reactor. The Sampler can be either screwed or snapped onto the Reactor. This RPS setting is intended for: storage of unused OMX-S every centrifugation process in-gel digestion. The Sampler (S) can also be attached on the Reactor (R) side. This SRP setting is intended for: operation of the Picker adding liquids into the Reactor through the Picker. 3

5 3. Detailed Protocol for the picking and tryptic in-gel digestion of protein spots 3.1. Required chemicals and laboratory apparatus (not included in the kit) Chemicals Water (Molecular Biology Grade, 18 megaohm or equivalent) Digestion buffer: Tris/HCl buffer (50 mm, ph 8.0) or ammonium hydrogencarbonate solution (50 mm, ph ca. 8.0) Trypsin stock solution: For tryptic in-gel digestion, 200 ng of modified trypsin (Promega/2005/Cat.- No.: V5111) in a maximal volume of 2 µl resuspension buffer (50 mm acetic acid; supplied by the manufacturer of the enzyme) is required per sample. Laboratory apparatus A clean, flat tray for washing the gel The tray size depends on the gel volume. Per cm 3 gel a volume of at least 20 ml water is recommended. A clean glass plate A light table (optional) A clean bench top micro centrifuge suitable for 1.5 ml standard tubes The centrifugation parameters are in units of relative centrifugal force (rcf). Pipettes and tips (designed for pipetting volumes of 18 µl and 2 µl, respectively) Thermomixer suitable for 1.5 ml standard tubes A temperature of 50 C and a mixing-speed of 1000 rp m are recommended for digestion procedure Procedure To efficiently use OMX-S for tryptic in-gel digestion we provide a step-by-step protocol for the analysis of protein bands or spots from 1D and 2D-SDS-PAGE-gels. It is recommended to stain proteins with soluble or colloidal Coomassie blue dye. For efficient digestion and high peptide recovery, it is essential not to fix stained proteins within the gel matrix using e.g. formaldehyde or glutaraldehyde. After OMX protocol, it is recommended to desalt and concentrate the resulting peptide solution on micro RP-silica tips. In case of direct use for LC-MS, please ensure that the solution contains no micro gel particles. 4

6 # Description Detailed instruction 1 Preparation of the gel Wash gel slab in water. 2 Drip off excessive water and transfer the gel to a cleaned glass plate. 3 Excision of spots Take an OMX-S, detach the Sampler from the Reactor, and put the Sampler on the bottom side of the Reactor. 4 Excise the protein band or spot of interest with the Picker. Please, refer to fig. A (p. 15) for details. 4a Repeat step 4 up to two times, if excision of bigger spots is intended. 5 Crush the gel Put the Sampler back on the picking side of the Reactor. Place the enclosed OMX-S in the centrifuge with the Reactor placed at the bottom of the centrifuge. Spin down gel. 6 Remove the OMX-S from the centrifuge and detach the Sampler. Put it on the bottom side of the Reactor and place the device in a rack. 7 Digestion of proteins Pipette 20 µl of digestion buffer containing 50 mm NH 4 HCO 3 (or 50 mm Tris/HCl) and 10 ng/µl of trypsin into the Picker. * 8 Place the enclosed OMX-S in the centrifuge. Please ensure that the Reactor is placed at the bottom of the centrifuge. Spin down the liquid. 9 Place the OMX-S in a thermo mixer with the Reactor positioned at the bottom of the thermo unit, and incubate under agitation (1000 rpm). 10 Separation of gel and analyte solution Place the enclosed OMX-S in the centrifuge. Please ensure that the Sampler is placed at the bottom of the centrifuge. Spin down the peptide solution. 2 x 5 min at RT 2 min at rcf x g 1 min at rcf 3800 x g 45 min at 50 C (Ref. 1, 2) 3 min at rcf x g * The usage of Brand pipette tips (2005/Cat.-No ) is recommended. If addition of higher volumes is intended, please provide liquid stepwise and centrifuge between steps or use geloader tips from Eppendorf (2005/Cat.-No ) to pipette the liquid directly into the Reactor. Maximal loading capacity of the Reactor is 40 µl. 5

7 Flow Chart Step no. according to step-by-step protocol from page 5: (3-4) (5-8) (9) (10) Fig. A: Excision of the protein band or spot of interest with the Picker 6

8 4. Troubleshooting Problem Step Possible cause Solution Gel stays pinned in the picker after centrifugation 5 Centrifugal force too low Incomplete digestion 1 Incorrect ph-value because of insufficient gel washing Low volume yield of peptide solution after post-digestion centrifugation Gel particles in the peptide solution after post-digestion centrifugation Higher rcf value or longer centrifugation time Wash the gel intensely with water before cutting the spot 7 Incorrect ph-value of reagents Ensure that ph of buffer is ~ Low enzyme activity Use a new Trypsin aliquot 11 Centrifugal force too Higher rcf value or the low centrifugation time 11 Centrifugal force too high Use rcf value and centrifugation time recommended in the protocol 5. Literature Havlis, J. et al., Anal Chem., 75(6), (2003) Finehout, E. J. et al., Proteomics, 5(9), (2005) OMX-S is a registered trademark of OMX GmbH. Coomassie is a registered trademark of ICI PLC. Eppendorf is a registered trademark of Eppendorf-Netheler-Hinz GmbH. 7