Western Blotting Systems CAT NO:EPS-B0015 & EPS-B0016
TABLE OF CONTENTS Important User Information Section 1 General Information 1.1 Introduction 1.2 Standard supply 1.3 Specifications Section 2 Instructions for Use 2.1 Cassette Set-up 2.2 Blotting Tank Set-up 2.3 Gel-Blot Transfer 2.4 Important Tips Section 3 Maintenance of Equipment 3.1 Care and Handling 3.2 Maintenance
IMPORTANT USER INFORMATION This Instruction Manual will explain how to use this product safely and effectively. Please read and carefully follow the instruction manual in its entirety. The triangle/lighting bolt symbol alerts the user of the product to potentially hazardous electrical exposure. Always turn off the DC power source prior to disconnecting power cords from the product. Disconnect power cords from the power source first and then from the product. For maximum safety, always operate this system in an isolated, low traffic area, not accessible to unauthorized personnel. Never operate damaged or leaking equipment Section 1 General Information 1.1 Introduction Western Blotting is a procedure used to transfer the electrophoretically resolved proteins to an immobilizing paper or membrane. This becomes a standard procedure for the sensitive and specific detection / identification of specific proteins. 1.2 Standard supply Main unit (electrophoresis chamber) and lid. Gel Cassette Electrode cassette Scotch brite pad Cushion pad Power cord.
1.3 Specifications Constructions: Buffer chamber, safety cover Acrylic Electrodes Platinum wire 0.2mm diameter. Cassette Acrylic Power cords 7500VDC, 500mA, 65 C Section 2 Instructions for Use 2.1 Cassette Set-up A sandwich should be made of the polyacrylamide gel as under- and over- layered with Scotch Brite pad, Sponge, 2 pieces of blotting papers and nitrocellulose membrane in order as mentioned in the figure.
Take the nitrocellulose membrane and soak it in trans-blot buffer for 5-10 minutes. Also soak the blotting paper layer (Whatman No.1) separately in the trans-blot buffer. Now place the pre-wetted nitrocellulose membrane on the wetted blotting paper (Whatman No.1) and then position the nitrocellulose paper directly on top of the gel. Pack this gel/nitrocellulose sandwich set-up inside the cassette. Ensure that the polyacrylamide gel of size (8X7 cm)should face the RED marked side of the cassette and the nitrocellulose membrane towards the BLACK marked side of the cassette. After positioning everything in the right place, fasten the cassette in a gentle manner. 2.2 Blotting Tank Set-up Place the gel/nitrocellulose membrane sandwich cassette into the transblot chamber of the blotting tank. Then place the electrodes in the blotting tank at its appropriate position and fill the tank with trans-blot buffer. The electrodes should get completely immersed in the buffer. Now gently place the cassette which should be pre-filled with trans-blot buffer. While placing the cassette in the trans-blot chamber, keep the RED marked side of the gel cassette facing the Positive terminal and the BLACK marked side of the cassette towards the Negative terminal. Before connecting the packed blotting tank to the current circuit ensure that the entire gel/nitrocellulose paper sandwich set-up should get soaked with the trans-blot buffer. 2.3 Gel-Blot Transfer Now connect the blotting tank to the High Current Power supply. Set the power pack s parameters as follow. Keep the Current (as constant) at 100mA and Voltage at 40V for 45 minutes -1 hour.
In an hour or so turn off the power supply after ensuring that the protein got transferred to the nitrocellulose membrane. Remove the cassette from the tank and dismantle the sandwich set-up. Separate the gel and place it in Ponceau s stain, in order to confirm the transfer of protein to the membrane. 2.4 Important Tips Take extreme care to avoid air bubbles while adding each layer. Air bubbles trapped in between the layers can be removed by rolling pipette or test tube gently over the layer. To achieve best results, strictly follow the steps mentioned in this manual. Changes in procedures may result in high background or poor band development. Be sure to wear rubber gloves rinsed with deionized water while handling gel and nitrocellulose membrane etc., Use clean containers and designate them for blotting purpose alone. Make sure that the size of the container permits free movement of the membrane during shaking and complete immersion in solution. Do not touch the membrane with bare hands or metal objects and does not put pressure on membrane while handling or changing solutions. All incubations should be done in glass dishes, not on plastic trays. SECTION 3 Maintenance of Equipment 3.1 Care and Handling The plastic components of the western blott are fabricated from acrylic. Electrodes and connectors are made from pure platinum, stainless steel. As with any laboratory instrument, adequate care ensures consistent and reliable performance.
After each use, rinse buffer chamber, gel tray and combs with de-ionized water. Wipe dry with a soft cloth or paper towel, or allow to air dry. Whenever necessary, all components may be washed gently with water and a non-abrasive detergent, and rinsed and dried as above. Never use abrasive cleaners, glass cleaning sprays or scouring pads to clean the components, as these will damage the unit and components. Additional precautions: Do not autoclave or dry-heat sterilize the apparatus or components. Do not expose the apparatus or components to phenol, acetone, benzene, halogenated hydrocarbon solvents or alcohols. Avoid prolonged exposure of the apparatus or components to UV light. 3.2 Maintenance The following inspection and maintenance procedures will help maintain the safety and reliable performance of the western blott systems. Replacement parts can be ordered by calling 044-24363199 or by contacting your local distributor. Banana plugs and power cords should be inspected regularly. If the banana plugs become loose or do not feel friction tight replace the plugs or power cords. Should power cord assemblies (connectors, wire or shrouds) show any signs of wear or damage (e.g. cracks, nicks, abrasions, or melted insulation), replace them immediately. The platinum wire is secured to the banana jack by compression between a stainless washer and the jack nut. The nut/washer interface should be tight and free of corrosion.