Proteomics Equipment Protocols

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Peter Elliott
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Bio-Rad Trans-Blot Cell Bio-Rad Protean 2D cell Prepared by: Bob Morrison

1 BioRad Protean IEF Cell BioRad PowerPac HC BioRad Reagents, precast Gels Proteomics Equipment Protocols BioRad/UMAX GS-800 Calibrated Densitometer Bio-Rad Trans-Blot Cell Bio-Rad Protean 2D cell Prepared by: Bob Morrison STLCC-CPLS, Instrumentation Specialist Dec 08, rev Oct 11 (links only) Page 1

2 Proteomics: Workflow- Typical (from STEMtech presentation) Nov 2010 Page 2

3 Proteomics : BioRad 2-D Systems Overview Link to BioRad IEF Methods and Products Manual (pdf) BioRad PowerPac HC Page 3

4 Proteomics : BioRad Protean IEF Cell, Basic Features Link to BioRad IEF Cell Instruction Manual (pdf) The PROTEAN IEF cell is a dedicated instrument for running IPG strips. High-capacity running platform accommodates up to twenty-four 7 cm strips or twelve 11, 17, 18, or 24 cm strips to increase efficiency and throughput Precision temperature control with a Peltier-cooled platform ensures reproducible focusing and an expanded operating range of C Integrated 10,000 V, 2.4 ma power supply helps achieve the desired volt-hours faster when maximum voltage is reached Disposable rehydration/equilibration trays simplify the transfer of rehydrated strips into the focusing tray, and have the added benefit of ensuring that residual sample buffer does not interfere with the current path through the strips and adversely affect results Tray options simplify handling. Each tray holds 1 12 IPG strips in individual numbered channels. For a fully automated approach, rehydrate and run in the focusing tray. To increase throughput, rehydrate strips in a rehydration/equilibration tray while running another set of IPG strips in the focusing tray in the cell Preset methods and real-time editing give maximum flexibility. The text-based user interface allows easy programming of methods and provides a continuous update of run progress Optional thermal printer records critical run data and provides a permanent record of the programmed method and all run events. Run data are collected via the RS-232 serial port Page 4

5 Proteomics : BioRad PowerPac HC Basic Features Link to BioRad PowerPac Instruction Manual (pdf) PowerPac HC power supply, with 3.0 A of current and 300 W of power. In addition to running standard horizontal agarose and vertical polyacrylamide gels, the PowerPac HC power supply easily handles all your blotting applications. To help you optimize every run, the PowerPac HC power supply provides fully adjustable voltage, current, and power controls. Runs 4 cells simultaneously Timer-controlled Fully programmable CE and EN safety compliant LCD screen shows all parameters during run Case is stackable Page 5

The compact size requires only 1 L of running buffer.

6 Proteomics : BioRad Criterion Cell Details Link to BioRad Criterion Instruction Manual (pdf) The Criterion cell was specifically designed to accommodate the innovative Criterion precast gel cassette. The compact size requires only 1 L of running buffer. The Criterion cell lid features a built-in wedge that opens Criterion cassettes or gel cassettes easily and consistently in one simple step. Criterion 135BR, P , SN , T091808BS, Cat Page 6

7 Proteomics : BioRad Trans-Blot Electrophoretic Transfer Cell Link to BioRad Trans-Blot Electrophoretic Transfer Cell Instruction Manual (pdf) The Trans-Blot cell is a truly versatile tank transfer system. Flexible electrode placement and variable power conditions allow standard or highintensity transfers of up to three 16 x 20 cm gels or up to twelve mini format gels simultaneously. Compatible with large and small gels from a variety of vertical electrophoresis systems depending on electrode placement, accommodates 1 3 large (16 x 20 cm) gels, 3 6 Criterion gels, or 4 12 Mini-PROTEAN or Ready Gel gels Electrodes may be positioned 4 cm or 8 cm apart Flexible electrode placement and variable power setting combine to allow standard field (30 V), overnight transfers or high-intensity field (200 V) transfers for rapid, hour-long experiments. A one-piece molded tank that uses just 2.5 L of buffer 3 color-coded gel holder cassettes, each with an effective blotting area of 16 x 20 cm, and a complete set of fiber pads Plate electrodes, consisting of platinum-coated titanium for the anode and stainless steel for the cathode, or a platinum wire electrode pair Super cooling coil 1 package blot absorbent filter paper (15 x 20 cm) Page 7

8 Proteomics : BioRad Protean II xi Cell The PROTEAN II xi cell is used in combination with the 16 x 16 cm gels, while the PROTEAN II XL wide format cell is used to perform 2-D separations with the 18.3 x 19.3 cm gels, which accommodate 17 cm IPG strips. The PROTEAN II xi and XL multi-cells, with six-gel capacity, ensure reproducibility for high-volume electrophoresis laboratories. Compatible transfer cells for the large PROTEAN II gels include the Trans-Blot cell and the Trans-Blot SD semi-dry cell. Link to BioRad Protean II xi Cell Instruction Manual (pdf) Page 8

9 Proteomics: ReadyPrep 2-D Starter Kit Instructions Link to BioRad ReadyPrep 2-D Instruction Manual (pdf) Page 9

10 Proteomics : BioRad, Equipment, Gels, Consumables Page 10

11 Proteomics: ProteanII Remove Comb & Clamps Remove Comb carefully from one end Remove sandwich clamp from end while holding plate Page 11

12 Proteomics: ProteanII Remove Clamps and Rinse Remove other sandwich clamp Thoroughly rinse edge of wells with DI water. Page 12

13 Proteomics: ProteanII Remount clamps Remount plates in sandwich clamps and lower assembly into upper slots first While pressing down and forward lock sandwich clamps into fixture Page 13

will be facing in toward the fixture center.

14 Proteomics: ProteanII Load plates to Cell Install other plate on sandwich clamps, ensure that the indented edge will be facing in toward the fixture center. Load both plates with sandwich clamps into cell center fixture. Page 14

cell Fill outer wells with buffer to a level of

15 Proteomics: ProteanII Fill outer wells with Buffer Load the entire sandwich clamp assembly into the cell Fill outer wells with buffer to a level of about one half full (must cover lower plate edge) Page 15

16 Proteomics: ProteanII Fill inner well with Buffer Fill the inner well (between the plates) completely full of buffer Approximate Fill level for buffer in outer wells Page 16

17 Proteomics: BioRad Protean II xi 2-D Cell Major Parts Page 17

18 Proteomics: Protean II xi Assembly Guide- Steps 1, 2 Page 18

19 Proteomics: Protean II xi Assembly Guide- Steps 3-5 Page 19

20 Proteomics: Protean II xi Assembly Guide- Steps 6, 7 Page 20

21 Proteomics: Protean II xi Assembly Guide- Steps 8, 9 Page 21

22 Proteomics: Protean II xi Assembly Guide- Step 10 & Multiple Gels Page 22

23 Proteomics: ReadyPrep2D, Imaging on Densiometer Page 23

24 Proteomics: Trans-Blot Cell Assembly Page 24

25 Proteomics: Trans-Blot- Prepare for Blotting, Steps 1-3 Page 25

26 Proteomics: Trans-Blot, Preparation, Steps 4-6 Page 26

27 Proteomics: Trans-Blot, Preparation, Steps 7-9 Page 27

Electrophoresis and Blotting: Isoelectric Focusing

Electrophoresis and Blotting: Isoelectric Focusing PROTEAN i12 IEF System 120 ReadyPrep Reagents for IEF 123 Tube Gel IEF 2-D Systems 124 Mini Format Analytical IEF 125 Ordering Information 126 IEF Systems