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