To design a Standard Operating Procedure that describes the procedure for operation, maintenance of SDS- PAGE Electrophoretic Assembly.
2.0 SCOPE:
This Standard Operating Procedure is applicable for the operation, maintenance and calibration of for SDS- PAGE Electrophoretic Assembly for biology department
3.0 RESPONSIBILITY
3.1 All personnel in the biology department shall be responsible for implementation and compliance to the SOP.
3.2 Head department shall be responsible to ensure the implementation and compliance to their SOP.
4.0 DISTRIBUTION
4.1 The Quality Assurance department responsible to keep SOP approved through proper channel.
4.2 The copy of ‘Control Copy’ of SOP is being circulated in the department and placed near allied Equipment.
5.0 DEFINITION (S) & ABBREVIATION
5.1 Definition
6.1.1 The equipment includes a casting stand and glass plates with connected bonded gel spacers that simplify hand casting and eliminate leaking during casting. The cell can run one to four gels, and the mini tank is compatible with other company electrode modules for tank blotting, 2-D electrophoresis, and electro elution.
5.2 Abbreviations
5.2.1 SOP : Standard Operating Procedure
5.2.2 SDS : Sodium Dodecyl sulfate
5.2.3 PAGE : Polyacrylamide gel electrophoresis
6.0 PROCEDURE
7.1 Introduction
An extremely normal strategy for isolating proteins by electrophoresis utilizes, polyacrylamide gel as a help medium and sodium dodecyl sulfate (SDS) to denature the proteins. The technique is known as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide gels confine larger molecules from migrating as fast as smaller molecules. Since the charge-to-mass proportion is almost something similar among SDS-denatured polypeptides, the last division of proteins is dependent generally on the differentiations in relative nuclear mass of polypeptides. In a gel of uniform thickness, the general relocation distance of a protein is adversely relative to the log of its mass.
7.2 Principle
Polyacrylamide gel electrophoresis separates molecules in complex mixtures according to size and charge. During electrophoresis there is a complex cooperation of tests, gel grid cushions, and electric flow bringing about isolated groups of individual particles. Subsequently the factors that should be considered in electrophoresis are gel pore size, gel cushion frameworks, and the properties of the atom of interest.
Gel pores are created by the cross linking of polyacrylamide with bis-acrylamide (bis) to create a network of pores. This structure allows the molecular sieving of molecules through the gel matrix. Gel pore size is a function of the acrylamide monomer concentration used.
7.3 Components
7.3.1 Spacer Plate
The spacer plate is the taller glass plate with permanently bonded gel spacers. Spacer plates are available in 0.75 mm, 1.0 mm, and 1.5 mm thicknesses, which are marked straight on each spacer plate.
7.3.2 Short Plate
The short plate is the shorter, flat glass plate that combines with the spacer plate to form the gel cassette sandwich.
7.3.3 Casting Frame
The casting frame, when placed on the benchtop, evenly aligns and captures the spacer plate and the short plate together to form the gel cassette sandwich prior to casting.
7.3.4 Gel Cassette Assembly
One casting frame, a spacer plate, and a short plate form one gel cassette assembly.
7.3.5 Casting Stand
The casting stand captures the gel cassette assembly during gel casting. It contains pressure levers that seal the gel cassette assembly against the casting gaskets.
7.3.6 Gel Cassette Sandwich
A spacer plate and short plate with polymerized gel form a gel sandwich.
7.3.7 Buffer Dam
The molded, one-piece buffer dam is used when running only 1 or 3 gels.
7.3.8 Electrode Assembly
The electrode assembly holds the gel sandwich. It lines the sealing gasket, the upper and lower electrodes, and the connecting banana plugs. The anode (lower electrode) banana plug is identified with a red marker and the cathode (upper electrode) banana plug with a black marker.
7.3.9 Companion Assembly
The companion assembly allows you to run gels 3 and 4. It holds the gel sandwich and houses the sealing gasket.
7.3.10 Mini Tank and Lid
The mini tank and lid associate to fully enclose the inner chamber during electrophoresis. The lid cannot be separated without disrupting the electrical circuit. The mini tank and lid are also well-matched with other Bio-Rad electrode modules for blotting, first-dimension of 2-D electrophoresis, and electro-elution.
7.4. Setup and Basic Operation
7.4.1 Gel Cassette Preparation
7.4.1.1 Place the casting frame upright with the pressure cams in the open position and facing forward on a flat surface.
7.4.1.2 Select a spacer plate of the desired gel thickness and place a short plate on top of it.
7.4.1.3 Turn the spacer plate so that the labeling is up. Slide the two glass plates into the casting frame, retaining the short plate facing the front of the frame (side with pressure cams). Note: Ensure that both plates are flush on a level surface and that the labels on the spacer plate are oriented correctly. Leaking may occur if the plates are misaligned or oriented incorrectly.
7.4.1.4 When the glass plates are in place, secure the pressure cams to secure the glass cassette sandwich in the casting frame (see Figure 3c). Check that both plates are flush at the bottom.
7.4.1.5 Place the casting frame into the casting stand by positioning the casting frame (with the locked pressure cams facing out) onto the casting gasket while engaging the spring-loaded lever of the casting stand onto the spacer plate (see Figure 3d). Note: The gray casting stand gaskets must be clean and dry. The casting stand gaskets are made of a special thermoplastic material that swells when soaked in water, so we recommend that you do not soak the gaskets for prolonged periods prior to casting. If the gaskets do get accidentally soaked and display swelling and/or deformation, just allow them to air dry and they will regain their original shape, size and performance.
7.4.2. Gel Casting
7.4.2.1 Discontinuous Polyacrylamide Gels
7.4.2.1.1 Place a comb completely into the assembled gel cassette.
7.4.2.1.2 Mark the glass plate 1 cm below the comb teeth.
7.4.2.1.3 Prepare the resolving gel monomer solution by combining all reagents except APS and TEMED.
7.4.2.1.4 Degas the solution under vacuum for at least 15 min. Do not use a sink water aspirator.
7.4.2.1.5 Add APS and TEMED to the degassed monomer solution and pour to the mark using a glass or
disposable plastic pipet. Pour the solution smoothly to prevent it from mixing with air.
7.4.2.1.6 Immediately overlay the monomer solution with water or t-amyl alcohol.
Note: If water is used, add it slowly and evenly to prevent mixing. Do not overlay with butanol or isobutanol.
7.4.2.1.7 Allow the gel to polymerize for 45 min to 1 hr. Rinse the gel surface completely with distilled water. Do not leave the alcohol overlay on the gel for more than 1 hr because it will dehydrate the top of the gel.
7.4.2.1.8 Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Degas under vacuum for at least 15 min.
7.4.2.1.9 Dry the top of the resolving gel with filter paper before pouring the stacking gel.
7.4.2.1.10 Add APS and TEMED to the degassed stacking gel monomer solution and pour the solution between the glass plates. Continue to pour until the top of the short plate is reached.
7.4.2.2 Continuous Polyacrylamide Gels
7.4.2.2.1Prepare the monomer solution by combining all reagents except the APS and the TEMED. Degas under vacuum for 15 min (refer to section 4 for gel formulations).
7.4.2.2.2Add APS and TEMED to the degassed monomer solution and pour the solution between the glass plates. Continue to pour until the top of the short plate is reached.
7.4.2.2.3Insert the desired comb between the spacers starting at the top of the spacer plate, making sure that the tabs at the ends of each comb are guided between the spacers. Seat the comb in the gel cassette by aligning the comb ridge with the top of the short plate.
7.4.2.2.4Rinse the casting frame(s) and stand with distilled, deionized water after use.
7.4.3 Electrophoresis Module Assembly and Sample Loading
7.4.3.1 Clean and dry Mini-PROTEAN Tetra cell tank.
7.4.3.2 Electrophoresis module (electrode assembly module only for 1 or 2 gels; for 3 or 4 gels also use the companion running module) • Running buffer (700 ml for 2 gels; 1000 ml for 4 gels).
7.4.3.3 Ready Gel precast gels or hand-cast gels.
7.4.3.4 Power Pack Basic power supply
7.4.3.5 When running 2 gels only, use the electrode assembly (the one with the banana plugs), not the companion running module (the one without the banana plugs). When running 4 gels, both the electrode assembly and the companion running module must be used, for a total of 4 gels (2 gels per assembly).
7.4.3.6 Set the clamping frame to the bare position on a clean flat surface (see Figure 4a).
7.4.3.7 Place the first gel sandwich or gel cassette (with the short plate facing inward) onto the gel supports; gel supports are molded into the bottom of the clamping frame assembly; there are two supports in each side of the assembly.
7.4.3.8 The gel will now rest at a 30° angle, tilting away from the center of the clamping frame. Please use caution when placing the first gel, making sure that the clamping frame remains balanced and does not tip over. Now, place the second gel on the other side of the clamping frame, again by resting the gel onto the supports. At this point there will be two gels resting at an angle, one on either side of the clamping frame, tilting away from the center of the frame (see Figure 4b).
7.4.3.9 It is critical that gel cassettes are placed into the clamping frame with the short plate facing inward. Also, the clamping frame requires 2 gels to create a functioning assembly. If an odd number of gels (1 or 3) is being run, you must use the buffer dam (see Figure 4b).
7.4.3.10 Using one hand, gently pull both gels towards each other, making sure that they rest firmly and squarely against the green gaskets that are built into the clamping frame; make certain that the short plates sit just below the notch at the top of the green gasket.
7.4.3.11 While gently squeezing the gel sandwiches or cassettes against the green gaskets with one hand (keeping constant pressure and both gels firmly held in place), slide the green arms of the clamping frame over the gels, locking them into place. Alternatively, you may choose to pick up the entire assembly with both hands, making sure that the gels do not shift, and simultaneously sliding both arms of the clamping frame into place (see Figure 4c).
7.4.3.12 The arms of the clamping frame push the short plates of each gel cassette up against the notch in the green gasket, creating a leak-proof seal (check again to make certain that the short plates sit just below the notch at the top of the green gasket). At this point, the sample wells can be washed out with running buffer, and sample can be loaded (Figure 4d).
7.4.3.13 If running more than 2 gels, repeat steps 1a–d with the companion running module.
Important Note: Do not attempt to lock the green arms of the clamping frame, without first ensuring that the gel cassettes are perfectly aligned and stabilized against the notches on the green gaskets of the module. To prevent the gels from shifting during the locking step, firmly and assigning grip them in place against the core of the module with one hand.
7.4.3.14 When running 1 or 2 gels only, do not place the companion run Module in the tank. Doing so will cause excessive heat generation and prevent electrophoretic separation.
7.4.4. Sample Loading
7.4.4.1 Fill the assembly (upper chamber) with buffer to just under the edge of the outer gel plate.
7.4.4.2 Load samples into each of the assemblies while they are sitting on a flat surface, outside of the tank.
7.4.4.3 Load the samples into the wells with a Hamilton syringe or a pipet using gel loading tips.
7.4.4.4 Insert the Hamilton syringe or pipet tip into the slots of the guide and fill the corresponding wells.
7.4.4.5 Load samples slowly to allow them to mend evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the syringe needle or pipet.
7.4.4.6 Samples may be loaded in the modules prior to placing the modules into the tank. Samples may also be loaded in the modules after the modules have been placed into the tank. Both methods will produce acceptable results. In both instances, the assembly (upper chamber) and the tank (lower chamber) should be filled with buffer.
7.4.5. Placement of the Electrode
Associations in the Mini-PROTEAN Tetra Tank Note: required total buffer volume, 700 ml for 2 gels; 1000 ml for 4 gels.
The Mini-PROTEAN Tetra tank has two positions in which to place two assemblies: the electrode assembly (back position) and the companion running module (front position).
7.4.5.1 Begin by placing the tank on a flat surface, with the front of the tank facing you (the front of the tank is the face that has the 2-Gels and 4-Gels line markings); when oriented properly, the red marking on the top inside edge of the tank will be on your right, and the black marking on the top inside edge of the tank will be on your left.
7.4.5.2 If running 2 gels only, you will be using just the electrode assembly, so place this assembly in the back position of the cell, making sure that the red (+) electrode jack matches the red marking on the top right inside edge of the tank.
7.4.5.3 If running 4 gels, place the electrode assembly (banana plugs) in the back place (as detailed in 2.2.3b.) and the companion running module (no banana plugs) in the front position. Make sure that in both instances the red (+) electrode is matching with the red marking on the top inside right edge of the tank. Note that incorrect orientation will not permit proper placement of the lid.
7.4.5.4 Fill the tank (lower chamber) with buffer to the indicated level (550 ml for 2 gels and 680 ml for 4 gels).
7.4.5.5 Place the lid on the Mini-PROTEAN Tetra tank. Make sure to align the color-coded banana plugs and jacks. The correct orientation is made by matching the jacks on the lid with the banana plugs on the electrode assembly. A stop on the lid prevents incorrect orientation.
Note that the raised tabs on each side of the tank will now slide through the slots in the lid, guiding the lid to a proper close. At this point, firmly, yet gently, press down on the lid with your thumbs using even pressure, till the lid is securely and tightly positioned on the tank.
Caution: When running 1 or 2 gels only, do not place the companion running module in the tank. Doing so will cause excessive heat generation and will prevent electrophoretic separation.
7.4.6. Power Conditions
7.4.6.1 Insert the electrical leads into a suitable power supply with the proper polarity.
7.4.6.2 Apply power to the Mini-PROTEAN Tetra cell and begin electrophoresis; 200 V constant is recommended for SDS-PAGE and most native gel applications. The same voltage (200 V) is used for both 2 and 4 gels. The optimal voltage for your application may differ. Run time is approximately 35 min* at 200 V for SDS-PAGE.
7.4.7 Gel Removal
7.4.7.1 After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.
7.4.7.2 Remove the tank lid and carefully lift out the electrode assemblies. Pour off and discard the running buffer. Note: Always pour off the buffer before opening the arms of the assembly, to avoid spilling the buffer.
7.4.7.3 Open the arms of the assembly and remove the gel cassettes.
7.4.7.4 Remove the gels from the gel cassette by gently separating the two plates of the gel cassette. Note: To remove the gel from a Ready Gel cassette, first slice the tape along the sides of the Ready Gel cassette where the inner glass plate meets the outer plastic plate.
7.4.7.5 Remove the gel by floating it off the plate by inverting the gel and plate under fixative or transfer solution, agitating gently until the gel separates from the plate.
7.5 Maintenance
7.5.1 Mini-PROTEAN Tetra tank and lid, rinse thoroughly with distilled water.
7.5.2 Glass plates and combs must wash with a laboratory detergent, then rinse thoroughly with distilled water.
8.0 PRECAUTIONS
8.1. Turn the power off before connecting the electrical leads.
8.2. Connect one lead at a time, using one hand only.
8.3. Ensure that hands are dry while connecting leads.
8.4. Keep the apparatus away from sinks or other water sources.
8.5. Turn off power before opening lid or reaching inside chamber.
8.6. Do not run electrophoresis equipment unattended.
8.7. If using ethidium bromide, have a hand-held UV light source available in the laboratory. Check working surfaces after each use.
8.8. Mix all stock solutions in a chemical fume hood.
8.9 Wear gloves while working.
9.0 REFERENCES & FORMAT
9.1 References
9.1.1 Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature 227, 680-685.
9.1.2 Ornstein L (1964). Disc electrophoresis. I. Background and theory. Ann N Y Acad Sci 121, 321-349.
9.1.3 Chrambach (1983). A and Jovin, T M, Electrophoresis, 4, 190–204 McLellan T (1982). Electrophoresis buffers for polyacrylamide gels at various ph. Anal Biochem 126, 94-99.
9.2 Format
9.2.1 Format – I Format for Log Book Maintenance
9.2.2 Format –II Format for Document History Sheet
10.0 REVISION HISTORY
Maintain the history of SOP or document as per the format. It shall be kept along with the current version of the SOP.
END OF THE DOCUMENT
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