BISC411
EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL
Examining T4 Lysozyme and Other Protein Production Using SDS Polyacrylamide Gel Electrophoresis
Note: Your group will make and run 2 gels..
A. Preparation of 14% Polyacrylamide Running Gel
Assemble "sandwiches" of two glass plates as demonstrated by the instructor.. Take notes during the demonstration. Do not press hard on the thin plates. They crack easily.
After the plates are assembled, add the following ingredients to a flask, in the order listed.
CAUTION: Unpolymerized acrylamide is a neurotoxin. Do not
mouth
pipet under any circumstances. Use dispensing
devices only. Once it has polymerized it is nontoxic, but it is still
a very good idea to wash your hands thoroughly with
soap and water if they come in contact with polyacrylamide.
Lower gel acrylamide solution 6 ml
10% ammonium persulfate
60 microliters
Mix by swirling the flask. Obtain a Pasteur pipet and bulb. Add 10 microliters of TEMED. Swirl gently to mix and dispense the solution immediately within the glass plates to within 3 cm of the top (to make room for the stacking gel). Avoid adding bubbles. You must pour this gel immediately!! Otherwise, the solution will begin to polymerize in the flask and you will be unable to pour it into the assembly.
As soon as the gel solution is cast, very carefully and slowly, add
water to the top of the gel with a pasteur pipette. Add
about 0.5 cm of water. Water will not mix with the denser acrylamide
solution but will compress it so that a sharp and flat
acrylamide gel boundary will be created as it polymerizes. Discard
the acrylamide solution left over in the marked
containers. Rinse the flask thoroughly with water and leave it to dry,
inverted on the work-bench on some paper towels.
Polymerization will take about 20 minutes.
After polymerization is complete, remove the overlay solution by inversion. Remove as much water as possible.
Mix the upper stacking gel solution by adding:
Upper acrylamide gel solution 3 ml
10% ammonium persulfate 30 microliters
Add 10 microliters of TEMED to the solution above. Swirl to mix and,
using a Pasteur pipet and a bulb, fill the space
between the plates atop the stacking gel. Avoid transferring
bubbles.
Immediately insert the Teflon comb slowly, directly into the
solution,
within the glass plates. Make sure the comb is straight. There should
be
a 1.5 to 2.5 cm space between the bottom of the wells and the
separating
gel. Be
sure no bubbles are present.
Polymerization will take about 30 minutes.
B. Loading and Running the Gel
Mark, with a marking pen, the location of the wells on the large
plate.
Carefully remove the white teflon comb which
forms the sample wells. Use upward, even, and gradual pressure
with your thumbs to lift the comb out without disturbing the wells.You
will see ten sample wells. Each sample well can accommodate up to
about 25 microliters of protein sample.
The instructor will then show you how to install the gel plates in
the
running apparatus. Take notes during the
procedure.
Add running buffer to the upper reservoir of the apparatus. Check for leaking. If there is none, proceed to add running buffer to the bottom until the bottom of the gel is completely submerged in buffer.
Carefully add 20 ul of each of your protein samples from the previous weeks to the wells (The order of the samples to be loaded will be given to you by the instructor). Also, load 10 microliters of the molecular weight ladder provided to you. Since the samples contains glycerol, they will sink and form a layer in the well.
Each protein sample must be delivered with a new pipettor tip.
Discard
used tips in appropriately labeled boxes on the
work benches. Do not dawdle while putting on the samples. Wells
sometimes
leak and samples will get cross-contaminated if you are slow.
Run these discontinuous slab gels at 200 volts with the power supply
regulation set at constant voltage. Runs take
about 40 minutes. Watch to see the blue dye front. Turn off the power
when it reaches the bottom of the gel.
When the power supply is ON, DO NOT TOUCH the apparatus (leads, fingers
in buffer tanks, etc….) under any
circumstance. Turn off everything and uncouple a lead if you have to
make any adjustment.
C. Removing Gels from Glass Plates, Staining and
Destaining
NOTE: There may be some alterations to this procedure. You
will be informed about this before class.
Remove the gel plates from the apparatus as demonstrated by the instructor.
Applying even, gentle, but steady pressure, pry the plates apart. This procedure requires skill and, if you're a beginner, some luck. It will be demonstrated and you will get help in class.When it works, the running gel and the stacking gel will remain intact. When it doesn't the gel is destroyed.
Using a spatula, scrape off the top stacking gel and wells by teasing it onto a paper towel. Dispose of it in the labeled biohazard bag. Put the plate and the running gel on the workbench.
Cut a small wedge on one side of the gel in the corner to orient where lane 1 and the top of the gel are.
Obtain staining solution in your plastic staining dish. Put the entire gel, still attached to the plate, into the staining solution. Be sure the gel is completely covered. Gently swirl the dish periodically, keeping an eye on the condition of the gel. If it begins to come off the plate that is fine but do not allow it to wrinkle up. Stain for 20 minutes.
Decant the stain into the designated bottle under the hood. Remove as much as possible. Be careful not to hurt the gel!! Cover the gel in destaining solution and again rock gently for 10 minutes. Decant again.
Repeat the destaining one more time.
One of the advantages of thin slab gels in that they destain quickly. You may destain again if needed. Once you can see the protein bands you may stop destaining, go to the gel documentation system and observe your results and take a picture of them. You can use your pictures to do the following measurements.
Measurements and Observations
Measure the distance travelled by each protein band in the molecular weight marker lane. Then measure the distance travelled by the T4 lysozyme samples and other proteins you can see. Use the molecular weights of the marker proteins (provided by the instructor) to construct a standard curve using semi-log paper as discussed previously. Use this curve to determine your experimentally measured size of theT4 lysozyme positive controls on your gels. Do they correspond to the size you found for T4 lysozyme when you researched it at the beginning of the semester?
Now, compare the lanes from the uninduced samples, unpurified
samples and the column
fractions. There should be a continuous smear of proteins in both the
uninduced and unpurified lanes from both the T4 lysozyme and other
protein cultures. Do you see a difference between the uninduced and
unpurified, indicating that the expression worked ? Can you see the
presence of any of these protein bands in any of your column fractions?
What does that indicate? Do your positive lysozyme assay fractions
correspond to what you see on your protein gels? Which is the
more sensitive method of determining the
fractions
that contain lysozyme, the activity assay or the gel analysis?
Does your total protein measurement from the biuret assays correspond
to what you see on the gels?