EXERCISE 8C Lab Procedures SAFETY WARNING Acrylamide in the unpolymerized form is a skin irritant and a potential neurotoxin Fortunately the acrylamide in your gels is polymerized so it should not present a safety hazard However as a precaution you are required to wear gloves when handling gels buffers stains and destaining solutions SAFETY WARNING Do NOT touch the electrophoresis chamber or wires when the voltage is turned on Voltages may be as high as 300 V and shocks can be fatal Before handling the electrophoresis apparatus always make sure it is disconnected from the power supply I Prepare the electrophoresis apparatus for SDS PAGE 1 Carefully study the diagram below It shows what your electrophoresis apparatus should look like when it is fully set up and ready to run General Diagram of Electrophoresis Set up with Vertical Gel Position electrodes connect red to red black to black well samples loaded here after area is flooded with appropriate buffer lid should fit snugly clamp holds gel in place during run base holds one of the two buffers resolving gel stacking gel line between resolving and stacking gels is subtle a lane the area under a single well This gel has ten lanes spacer between glass in front and metal behind gel between do not throw this away Diagram courtesy of Steve Bostic ACC faculty Exercise 8C Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 8C 7 2 With gloved hands carefully unwrap the cellophane from your electrophoresis gel Notice that the actual gel is sandwiched between a white metal plate and a glass plate Two gray plastic spacers are located on either side of the gel and a white plastic comb is sticking out from the top of the gel This comb was used to form the wells indentations in the top of the gel 3 Handle the gel sandwich gently to avoid distorting or breaking the gel Carefully remove the white plastic comb from the gel and drain any excess water out of the wells Wash and save the gel comb do NOT throw it away Now clamp the entire gel sandwich onto the electrophoresis apparatus with the metal plate pushed firmly against the rubber gasket 4 Place a clear plastic template on the glass plate and align the black lines with the wells in the gel This will allow you to locate the wells once they have been filled with cathode buffer 5 Use a Pasteur pipette to fill the wells with cathode buffer Force the liquid into the wells with some velocity in order to sweep any non polymerized gel components out of the wells No harm is done if some Cathode Buffer splashes or overflows into the top reservoir the narrow space behind the gel support chamber since this reservoir will be filled with the same solution 6 Pour cathode buffer into the top reservoir until the liquid level is 3 4 mm below the top of the glass plate This is necessary in order to make electrical contact between the electrode and the gel 7 Check to make sure that the cathode buffer is not leaking down from the upper buffer chamber If it is leaking add vacuum grease to the rubber seal and adjust your clamps to stop the leak before continuing with the experiment II Prepare your samples and load them into the wells of your gel 1 Last week you saved 8 Eppendorf tubes containing samples of your milk fractions with 2X sample treatment buffer added These tubes have been placed in an ice bucket along with an additional Eppendorf tube containing a mixture of Molecular Weight Standards MW 2 If you need to dilute any of your samples before loading them into your gel make your dilutions now Make your dilutions according to the table below which you completed as part of the Prelab Dilution of Milk Fractions to Make 40 L of Diluted Sample with a Protein Concentration of 1 25 g L Milk fraction Amount of milk fraction with 2X sample treatment buffer needed L Amount of 1X sample treatment buffer needed L Skim milk Pellet Whey Column fraction Column fraction Column fraction Column fraction Column fraction Exercise 8C Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 8C 8 3 Insert all 9 Eppendorf tubes milk fractions plus MW standards into a holder and place them in the 60 C water bath Make sure you use the diluted samples for those fractions that required dilution and the undiluted samples for those fractions that did not require dilution Heat the samples for 5 minutes removing them briefly at 1 minute intervals to mix by tapping the sides of the tubes After 5 minutes dry the Eppendorf tubes and place them in the microcentrifuge in balanced positions Centrifuge for 5 sec to force the solutions to the bottoms 4 Return the Eppendorf tubes to the ice bucket and leave them undisturbed to allow any aggregated proteins and insoluble particulate matter to settle 5 Your gel contains 10 wells The wells are identified as 1 through 10 starting with 1 on your left as you face the front of the gel Following the instructions below load 16 L of sample into each well Important Load your samples carefully to make sure that each sample stays in its proper well Ultrafine micropipette tip should be gently pressed against glass front and held near top of well Sample should fall gently into bottom of well and be clearly visible Do not puncture gel Area should already be flooded with appropriate buffer Volumes to be loaded should be calculated in advance well Loading Well in Vertical Gel clamp Diagram courtesy of Steve Bostic ACC biology faculty To load your first sample adjust an automatic pipettor to deliver 16 L and attach an ultra thin disposable capillary tip Withdraw the correct amount of your milk sample from your Eppendorf tube labeled Milk and insert the tip of the pipettor into the top of well 1 Make sure that the pipettor tip is between the glass and metal plates of the gel sandwich Keep the tip at least 4mm above the bottom of the well Take care not to puncture the sides or the bottom of the well with your pipettor tip This takes a steady hand it may help to support the micropipettor with your other hand and to support your elbows on the lab bench top Very slowly and gently expel the solution from the pipettor tip into the well while holding the pipettor steady The blue solution should fall to the bottom of the well gradually filling it Do not press the pipettor to the second stop it is important to avoid blowing air bubbles into the well Do not release your thumb from the pipettor until you have completely withdrawn the tip from the well If the sample overflows into an adjacent well you may