SDS-PAGE Separation of Proteins Produced by Beans During Development
Background Information
Sodium Dodecylsulfate (SDS, also called Sodium Lauryl Sulfate) is an anionic detergent
which denatures proteins by wrapping around the peptide backbone. This results in two things 1)
a net negative charge proportional to the length of the protein in question and 2) uniform rod
shaped proteins. Because shape is not a factor, as all proteins exposed to SDS will be rod shaped
and charge is proportional to the length of each protein, it is possible to separate these proteins on
the basis of size using gel electrophoresis.
In gel electrophoresis, molecules with a net electrical charge, in this case a negative
charge due to the SDS, are placed in an electrical field. Under these conditions, the charged
molecule moves toward the pole with a charge opposite to that carried on the molecule. In this
particular case, the proteins move toward the positive pole as they carry a negative charge.
Usually the molecules are forced to move through a gel matrix. As smaller molecules can move
through the matrix faster than large molecules, the molecules become separated with fast moving
bands of small molecules at the front and slow moving bands of larger molecules trailing behind.
Proteins produced during the process of seed germination in beans will be separated using
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE in this laboratory). Beans will be
allowed to germinate for different periods of time ranging from zero to ten days. They will then
be ground up and the proteins extracted. The proteins will be separated on a polyacrylamide gel
matrix.
Polyacrylimide is a polymer of acrylamide and N,N’-methylene-bis-acrylamide (bis). The
bis provides the crosslinks between long polymers of acrylamide. As a consequence the pore
size of the gel can be controlled by varying the ratio of acrylamide to bis. Typically, a ratio of
37.5:1 of acylamide to bis is used to separate proteins although it may be altered depending on
the size of the proteins being separated.
Preparation of Germinated Beans
Beans will be started germinating 10 days before the lab and every day that follows, more
beans will be started so that by the time lab is held beans will have been germinating for 10, 9, 8,
7, 6, 5, 4, 3, 2, 1, and 0 days. Bean germination will be achieved by placing a wet paper towel in
a glass culture dish then placing 6 - 8 beans on the towel. After placing another wet paper towel
on top of the beans, a second culture dish should be placed on top of the first one to prevent
excessive evaporation. Be sure to carefully mark each dish with the date that the seeds were
started germinating.
Preparation of the Polyacrylamide Gel
Because acrylamide is a dangerous neurotoxin and is also a suspected carcinogen,
great care should be exercised in its handling. Always wear gloves, and clean up spills
immediately. Do not eat or drink anything in lab, or before washing your hands after lab. You
will be using a premixed acrylamide solution that is not quite as dangerous as the dry powder, but
should still be treated with caution
The type of gel that we will be casting is a discontinuous gel. In this type of gel, the part
of the gel used for separation of the fragments is cast first, then a second gel called a stacking gel
is cast on top. The staking gel causes proteins in dilute solutions to form a very sharp
concentrated band before contacting the separating gel. This allows the loading of dilute protein
solutions onto the gel while still producing sharp bands on the separating gel.
Casting the separating gel
1
Make sure that all parts of the vertical gel electrophoresis chamber are absolutely clean
then assemble the parts for gel casting.
2
Measure the volume of the space in which you will be casting the separating gel.
Remember to leave 3 cm at the top of the gel for the stacking gel.
3
We will be casting a 7.5 % acrylamide gel, so the following calculations must be made:
7.5g 100ml
x
xXml = Yml
100ml 40g
Volume of 40 % acrylamide solution.
Where X is the volume of the gel, and Y is the volume of 40 % acrylamide
solution to use.
Volume of 2 x Glycine SDS Buffer.
50 % of the final volume should be made up using 2 x Glycine buffer solution
containing SDS.
Volume of 10 % Ammonium Persulfate
0.5% of the final volume.
Volume of TEMED
0.033 % of the final volume.
Volume of Distilled Water
The difference between what has already been calculated and the final volume.
4
Once all the volumes have been calculated, all the components of the gel should be
combined except for the TEMED and the Ammonium persulfate. When you have double
checked and read ahead the next few steps, the TEMED and Ammonium persulfate
should be added and the solution throughly mixed, but not violently shaken.
5
Once the components of the gel are mixed, pour them into the gel sandwich, then
immediately overlay the gel with 2 ml of distilled water.
6
Leave the gel to set for at least 30 min. Then proceed to casting the stacking gel.
Casting the stacking gel
1
The staking gel will be a 4 % polyacrylamide gel. Calculate the volumes necessary to
make the stacking gel with this final make up, but adjust the buffer to be used to pH 6.8
using either HCl or NaOH.
2
Combine all components as you did in the separating gel, then mix throughly and add the
solution to the top of the separating gel.
3
Immediately place the Teflon comb. in the top of the staking gel to form wells.
4
Let the gel set up for at least 60 min.
While the stacking gel is hardening, proceed on to the steps involved in preparing the
proteins for separation.
Extraction of Proteins and Preparation for SDS-PAGE
Obtain the germinated beans that you have prepared over the past twelve days. Before
anything else is done, a careful record should be made of the appearance and weight of beans
following each germination period. As far as is possible, seeds of the same mass and size should
be selected from each culture dish for comparison. Add one seed from the 12 day germination
culture dish to a 50 ml test tube containing 5 ml of Protein Solublization Solution (62 mM TrisHCl, pH 6.8, 10 % glycerol, 2% SDS, 5 % β-mercaptoethanol, and 0.00125 % bromphenol blue).
Grind the seed for approximately 15 seconds in the polytron. Transfer 1 ml of the solution to a
1.5 ml microcentrifuge tube and centrifuge at high speed for 30 seconds to precipitate any large
chunks. Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube and place that tube into a
boiling water bath for 4 minutes. This last step heat-denatures any proteins that remained folded
during earlier steps so that SDS can get to their entire length. Allow the tubes to cool to room
temperature and they are ready to be loaded onto the gel.
Electrophoresis
Rearrange the vertical gel electrophoresis apparatus for electrophoresis. Remove the well
forming comb from the top of the stacking gel. Add the appropriate protein solutions (20 μl) to
each well. Fill the upper and lower buffer chambers of the gel setup with running buffer. Attach
electrical leads to the power supply and switch to constant current. Adjust to 1.5 mA/cm of gel
width. The protein’s passage through the gel can be followed by watching the blue tracking dye
as it travels down the gel. When the tracking dye has reached approximately 2.5 cm from the
bottom of the gel, the power should be switched off and the gel stained. Time to travel through
the gel should be proximately 20 min for every 3 cm of gel length.
Gel staining
Take apart the electrophoresis set up and place the gel in a flat bottomed container. Add
commassi blue stain and leave the gel to stain over night. In the morning, pour off the stain
solution and add approximately 200 ml of destaining solution (to make 1 l combine 200 ml of
glacial acetic acid, 300 ml of methanol, 600 ml of distilled water). The destaining solution
should be changed every 30 min until parts of the gel lacking protein are clear, while the proteins
remain blue. Excessive destaining will result in loss of band visibility.