SDS-Polyacrylamide Gel Electrophoresis

It is an annual battle for us to perform the gel electrophoresis DEMO because only rarely has a TA prior experience in this method. The purpose of this document is to line out some of the things we need and set up a schedule that will lead to success with minimal stress. .

About TWO WEEKS prior to the lab, the TA should begin rounding up and testing the following equipment.

# WHAT? WHERE IS IT? COMMENTS SOURCE PICTURE

1 Miniprotean apparatus Lab 138 Gel Electro. We have XXX Biorad Drawer of these units. #XXXXXX 2 Power Supply & Lab 138 Gel Electro. We have XXX Biorad connecting leads Drawer of these units #XXXXXX 3 SDS Running Buffer Lab 138 Gel Electro. It will be diluted Biorad Concentrate, Drawer by ten, so lasts a #XXXXXX Bio-Rad XXXXXX fairly long time. 4 Laemmli Buffer Lab 138 Gel Electro. Contains Biorad Drawer bromophenyl #XXXXXX blue to indicate leading edge of each lane, some other chemicals. 5 2-mercaptoethanol Borrow from friends—e.g., You only need Biorad (a.k.a.  Waldrop or Licata tiny amounts, so #XXXXXX mercaptoethanol or borrow this ME) smelly stuff. 6 Marker proteins Borrow from friends—but It would be Biorad we replenish these for them smart to buy #XXXXXX (their incentive to ahead of the lab contribute). Stuff doesn’t and give the last for a whole year remainder to our anyway. friends afterwards. 7 Coomassie blue Lab 138 GelElectro. XXXXNeed to Biorad Drawer order this. #XXXXXX 8 Eppendorf centrifuge Lab 138 GelElectro. tubes Drawer 9 Ethanol or Methanol Get from organic Need about 500 stockroom? mL 10 Glacial acetic acid Get from organic Need about 100 stockroom? mL 11 Test proteins LAB 230 Refrigerator BSA and other assorted proteins 12 Prefab gels LAB 230 Refrigerator Ready-made Biorad PAA gels in #XXXXXX cassettes 13 Staining Solution (Coomassie blue in alcohol/water/acetic acid)

If we cannot borrow some, here’s a recipe from the Stephen Hand lab:  250ml isopropanol  100ml acetic acid  2.5g coomassie blue R-250  Dilute with nanopure water to 1L  Store at room temp.

SDS Running Buffer

 Just dilute 100 mL of the concentrate (item #3) to 1L total using deionized or nanopure water.

 If we don’t have the running buffer concentrate from BioRad, start-from-scratch procedures are found on links near to the one that opened this file. Such a start-from scratch procedure is only marginally illustrative in 4010 class, but it saves $$$ when many students are enrolled.

BASIC PROCEDURE

Make the Denaturant Solution

 To an Eppendorf centrifuge tube, add 950 mL of blue Laemmli buffer and 50 mL of 2- mercaptoethanol (a.k.a. ME).

Denature the Test Proteins

 Dissolve the test protein to approximately 1 mg/mL.

 To an Eppendorf centrifuge tube, add 10 mL of the test protein solution and 10 mL of the denaturant solution.

 Heat in a water bath to approximately 80ºC for about 15 minutes (the temp and time is just a guess, but seems similar to other procedures).

Denature the Marker Proteins

 Do while test proteins are cooking.

 To an Eppendorf centrifuge tube, add 10 mL of the marker solution and 10 mL of the denaturant solution.  Heat in the same water bath as test proteins. The time will be almost the same (i.e., if it took you one minute to mix the marker proteins with the denaturant, cook the markers an extra minute).

Assemble the device following the BioRad manual. Here’s what matters most:

 You should have practiced before ever starting any of this.

 Do while marker and test proteins are cooking.

 Make sure the LOWER glass plate of the gel cassette faces inward towards the center of the device.

 If using a plastic dam instead of a second cassette, make sure to follow the orientation marks indicated on the plastic dam. (This side towards gasket)

 Make sure the seal is good between the glass plate cassette(s) and dam (if used) and the green gasket.

 You can test the above two criteria by pouring some running buffer (or just water) into the volume between the two cassettes (or cassette and dam). It should not leak out. If you used water instead of running buffer, pour it out.

 The inside water level must be above the lower (inner) glass of the cassette and below the upper (outer) glass. This allows the electrical circuit to be completed.

Load and run the Samples and Marker Proteins

 Remove the comb CAREFULLY. Pull it straight out.

 Try not to disturb the gel comb so your wells remain separated by gel protrusions.

 Add 10 ml of the marker protein to left most and rightmost lanes.

 Add 10 ml of test protein to several lanes (for reproducibility).

 Run at XXX V until the blue bromophenol dye has migrated almost to the bottom. This will be about XXX minutes.

TA Advance Preparation Timeline

TIME AHEAD WHAT OF LAB 2 weeks Work with the instruction manual on line and with the professor to learn how the device is assembled. Check inventory and order everything needed. Order everything needed.

1 week Talk to Waldrop or Licata for items we may need; tell them we will reimburse, as usual. Practice run. 1 day Set up