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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.
Solutions:
Loading Buffer
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
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.