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Lisa Jungbauer and Courtney Bakke, 05/30/03;

Tris-Tricine SDS-PAGE Gel for Peptides/Small Proteins


(good for Mb and the N-terminal fragments)
Pouring 16.5% Tricine 2-Layer Gels:
1. Clean glass plates (1 thick plate and 1 shorter thin plate per gel) with distilled
water and rinse with 70% Ethanol. Dry plates with a Kim-wipe make sure the
plates are dry before clamping.
2. Clamp plates into gel apparatus with the thin plate out - keep any chips on the
plates out and towards the top (to prevent leaking). Use a thin Sharpie marker to
mark on the glass plates where the gel layers should be.
3. Add a bead of the resolving gel to the bottom of the plates to prevent leaking
Add the resolving gel in between the plates and fill a couple millimeters over the
line as the plates leak slightly. Slowly pipette a layer of H2O on top of the
resolving gel about a centimeter thick. Wait until left over resolving gel has
solidified (~ 4560 min).
4. Get rid of water and any unpolymerized gel. Unclamp the top of the gel plates (do
not undo the green clamps!) and tip upside down in the sink. Hold the gels by the
green clamps so the plates dont shift. Wash in between the plates with the water
squirt bottle the use a Kim-wipe (capillary action) to dry. Wipe off the bottom
sponge to preserve it.
5. Clamp plates back into gel apparatus. Add stacking gel until it overflows. Push
the comb in even with the thick plate. Wait until extra stacking gel has solidified
in conical tube.
6. Can use the gels right away or can store the plates at 4C overnight. If storing
them overnight remove the gels from the green clamps but LEAVE THE
COMBS IN and place them in wet absorbent towels in a plastic bag. Use 1X gel
buffer to wet the towels.
7. If using the gels right away - pull out comb slowly and evenly, then remove the
green clamps and rinse wells and plates with a water squirt bottle.

Sample Preparation
1. Prepare samples in loading buffer
a. (example3 uL sample +17 uL loading buffer) Do not exceed a total
volume of 20 uL or sample could overflow into adjacent wells. Give
samples a quick spin before boiling.
2. Boil the samples and marker for 3 minutes then place on ice and centrifuge
briefly. Samples are now ready to load.
3. For Myoglobin (because it does not contain disulfide bridges) it is not
necessary to add beta-mercaptoethanol or DTT to the running buffer.
However, for the molecular weight standards, it is essential to add one of these
reducing agents.
4. Use Broad Range Molecular Weight Standards:
a. A good recipe: 18 uL loading buffer + 1 uL Markers + 1 uL betamercaptoethanolboil for 10 minutes
Notebe cafeful with BMEit STINKS! Add it in the hoodavoid
touching the pipet to the container or the pipet will stink for daysdispose of
the tip used in the container in the hood to avoid stinking up the bench.
Whatever you get the BME on will stink p.u. and no one will want you to
work in the lab.

Running the Gel:


1. Take out gel rig (2 pieces) place the thin plate towards the inside. If using
only one gel, must use a buffer dam on the other side. Keep pressure on the top
while closing the doors.
2. Place the gel rig into tank and fill the middle with 1X running buffer to the top
of the glass plates. Next fill the outside almost to the top of the doors
(approximately 1- 1 inches). Place the yellow loading guide in between the gels.
3. Add each of the samples using gel loading pipette tips. Slowly add sample into
the gel lanes. Try to avoid any air at the end of the tip and dont blow out tip all
the way to avoid bubbles.
4. Load 5-10 uL of the markers. Remove the guide bar when finished and put lid on
the tank.
5. Run gel at 100 volts for 100 minutes.

Solutions:
Loading Buffer

Running Buffer (10X)*

99 mg of Coomassie: G-250 (0.66%)


3 ml of glycerol (to weight down proteins)
0.6 g of SDS (helps redissolve pellet)
1.5 ml of 1M Tris pH 6.8
fill to 15 ml with ddH2O

60.55 g of Tris base


89.60 g of Tricine
5.0 g of SDS
fill to 500 ml with ddH2O

(or the solution in the BioRad Ready Gel


Applications guide on pg 12 can be used)

*10X is the stock solution want 1X to run gel


50 ml of 10X + 450 ml H2O = 500 ml of 1X

2-Layer Gels: Recipe for 2 gels


30% Acrylamide
1.5 M TrisCl, pH 8.8
1.0 M TrisCl, pH 6.8
ddH2O water
10% SDS (w/v)
10% APS (w/v; make fresh)
TEMED

Resolving Gel
5.5 ml
2.5 ml
----1.9 mL
0.1 mL
0.05 mL**
5 l**

Stacking Gel
830 l
-----630 L
3.4 mL
0.05 mL
0.05 mL**
5 l**

** Add right
before use

- make up solutions in 50 ml conical tubes


- carefully roll tubes to mix to avoid getting air bubbles

30% Acrylamide
Wear a mask when weighing out the acrylamide and bisacrylamidethey are
neurotoxins! (once dissolved into solution they are not as harmful)
Be careful when adding waterdont add the full amount initially because the
dissolution of the acrylamide causes volume expansion.
In 250 ml glass graduated cylinder add:
29.2 g Acrylamide
0.8 g NN-bis-methylene-acrylamide
Add 70 mL of H2O (from carboy) acrylamide increases the volume when it dissolves
Heat to 37 in water bath to dissolve with swirling to stir and help dissolveafter most
of the powder is in solution add ~ 10 mL more water (unless volume is already at 100mL)
to help the final bit dissolve.
Bring final volume to 100 ml
Filter and store at 4C in a foil-covered amber bottle (to prevent light damage)
*Date bottle and make fresh once every 1-3 months
10% APS
10 g APS / 1 ml H2O = 50 mg/500 l or 10 mg/100 l
*Make fresh every time
10% w/v SDS:
10 g SDS in 90 mL H2O, gentle stirring (to avoid foamy bubbles)
Bring final volume to 100 mL
1.5 M TrisCl, pH 8.8
27.23 g Tris Base in 80 mL dH2O
adjust pH to 8.8 with concentrated HCl
bring volume to 150 mL with dH2O
store at 4 deg C
1.0 M TrisCl, pH 6.8
24.23 g Tris Base in 110 mL dH2O
adjust pH to 6.8 with concentrated HCl
bring final volume to 200 mL with dH2O
store at 4 deg C

Gel Staining:
(see pg. 23 of BioRad Ready Gel Application Guide)
Peptides and small proteins are prone to diffusion and loss during staining. The
following protocol uses a fixation step to prevent sample loss and is suitable for detection
of bands as low as 10-20 ng.
Fixative solution
40% methanol (400 mL)
10% acetic acid (100 mL)
dH2O
(500 mL)
Coomassie Blue Staining Solution
0.025% Coomassie Blue G-250 (0.25 g)
10% acetic acid
(100 mL)
dH2O
(900 mL)
Destain Solution
10% acetic acid
dH2O

(100 mL)
(900 mL)

Place gels in fixative solution and equilibrate for 30 min on the gel rocker.
Stain gels with Coomassie Blue stain for 1 hour. Stain should only be used once.
Destain gels 3 x 15 minutes changing the solution between each stainput a kimwipe in
the container to help wick away dye molecules.
Or.. destain 1 x 15 minutes in destaining solution and then leave overnight in
dH2O with a Kimwipeshould be destained by morning.

Gel Drying:
The gel will be placed between two gel drying films (from Promega) and dried overnight
in the hood for permanent storage. Scan the gel before drying it in case the gel cracks or
tears during the drying process, and to have a source of permanent data for record in your
notebook.
One sheet from the box can be used for 1-2 gels, to conserve the expensive film.
Make sure to remove all air bubbles from between the sheets of the films or they will
cause cracking.
Gel Drying Solution:
40% methanol
10% glycerol
7.5% acetic acid
Place the gel in the drying solution for 3-5 minutes
Thoroughly moisten one sheet of gel drying film with the drying solution (~ 2 mL)
Place the gel on the wet film and position and remove air bubbles
Place more gel drying solution on top of the gel (~ 1 mL).
Place gel drying solution on the second piece of film (~2 mL) and flip it onto the gel,
again positioning and removing air bubbles
Clamp the gel in to the frame and store vertically in the hood until dry.

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