CHEM 3131
Fall 2014
Using Isoelectric Focusing Gels to Analyze Chemically Modified Proteins
Isoelectric focusing gels separate proteins based on the isoelectric point of the protein. The
isoelectric point of a protein is the pH at which the protein is neutral. Isoelectric focusing gels take
advantage of the fact that the proteins will sensitive to the pH of the environment, gaining or losing
protons in response to the environmental pH. If a protein is in a pH environment that is below the pI,
the protein will be positively charged. If the protein is in a pH environment that is higher than the pI,
the protein will be negatively charged. At the pH that is the pI, the protein is neutral.
The proteins, in the presence of an electric field, and a linear gradient of pH, migrate through
the gel initially toward the electrode of the opposite charge that they have. They continue to migrate
until they reach the pH that is equal to the pI. They migrate through the medium, in the case of the gels
used here, gaining protons (H+) as the proteins go toward the lower pH values. The protein then starts
to lose charge and slows down. Once the proteins are at the pH that is the pI of the protein, the protein
no longer has a charge and stops moving. If the protein happens to diffuse away and encounters a
lower pH, the protein will acquire a positive charge, and be forced toward the cathode by the electric
field. On the other hand, if the protein diffuses toward a higher pH value than its pI, the protein will
become negatively charged and drive toward the anode. This process allows for a sharp, focused band
to appear at the pI of the protein.
The gels could be made in two ways. First, the gels have the acrylamide, bisacrylamide and
ampholytes present. Ampholytes are collections of organic molecules that range between 300-1000 Da
that differ in the number of titrateable groups. In the presence of an electric field, these molecules
partition into a linear gradient of pH values. The second way that the pH gradient can be made is
through the use of immobilines. These are covalently linked acrylamide monomers and buffers. The
gels then are poured to have a gradient of pH values. The isoelectric focusing gels that are used in this
lab (Life technologies) are generated using a 5% acrylamide/ bisacrylamide with 2% ampholytes.
In this experiment, you will be given samples for three different proteins from the list below.
Your job is to identify these samples using bioinformatics and isoelectric focusing gels. You will
determining the theoretical isoelectric points of the proteins using bioinformatics and match the sample
to the theoretical pI based on how they run on the gel. In addition to the isoelectric point, a single band
versus multiple bands also tells us about how homogeneous the population is for each protein.
List of proteins:
1. Trypsin inhibitor B (source: Glycine max)
2. Myoglobin (source: Equine skeletal Muscle)
3. Carbonic anhydrase II (source: Bos taurus)
References cited
Garfin, David E. “Gel Electrophoresis of Proteins” Pg 197-268 in Essential Cell Biology, Vol 1 Davey, John
and Lord, Mike Eds., Oxford University Press: Oxford (2003)
Bjellqvist, Bengt et. al ”Isoelectric focusing in immobilized pH gradients: Principle, methodology, and
some application” (1982) J. Biomed. Biophys. Meth. 6, 317-339.
CHEM 3131
Fall 2014
Gel Analysis of Diverse Proteins
1. Remove samples from the -20oC freezer and let thaw.
2. You will need to run 3different samples on the IEF gel. Add 10 µL of your samples to 10 µL of
the IEF sample buffer. Mix well.
3. Each group will have 3 samples of different proteins to run. Two groups should run on 1 gel.
For each group, there will also be an IEF Marker.
4. There should be 2 gels of the pH 3-10 IEF gels (Life Technologies) run in each gel box. For each
gel box, open the 2 IEF gels. Remove from the bag and peel off the white strip. Take a marker
and indicate where each well is on the gel. This makes it much easier to load the gel.
5. Add both gels to form the upper chamber, with the wells facing to the center of the chamber.
Lock into place.
6. Obtain 200 mL of chilled IEF pH 3-10 cathode buffer. Fill the upper chamber with the chilled
cathode buffer. Check for leaks.
7. Obtain 600 mL of chilled IEF pH 3-10 anode buffer. Use about 300 mL to partly fill the outer
chamber.
8. Remove the lanes comb. Load 5 µL of IEF Marker in the far left lane. The first group then loads
their 3 samples; the second group then loads their 3 samples and a marker.
Lane 1: Group 1 IEF Marker
Lane 2: Group 1 Sample 1
Lane 3: Group 1 Sample 2
Lane 4: Group 1 Sample 3
Lane 5: Empty
Lane 6: Group 2 IEF Marker
Lane 7: Group 2 Sample 1
Lane 8: Group 2 Sample 2
Lane 9: Group 2 Sample 3
9. Once both gels have been loaded, fill the rest of the outer chamber with the remaining 300 mL
of chilled IEF pH 3-10 anode buffer.
10. Attach the lid to the gel box and connect to a high voltage power supply. Run at 100 V
(constant) for 1 hour. Run at 200 V for the next hour. Run at 500 V for the last 15-30 minutes.
11. Remove gels from the box. Put into 30 mL of “fixing” solution containing 12 % trichloroacetic
acid and 3.5% sulfosalicylic acid. “Fixing” the protein allows it to keep from diffusing after the
electric field is removed.
12. Remove the fixing solution and discard in the waste container. Rinse the container with a little
nanopure water and discard. Then, fill the staining box with ~60 mL coomassie blue staining
solution. Stain overnight. Be sure that the gels are flat, and that every portion of the gel is
equally exposed to the coomassie for best staining.
13. The next day, take out the staining solution. Rinse the box with a small amount of destain and
discard in the wast. Then fill with ~60 mL of destaining solution. Take a picture (see directions
for using Gel Doc EZ Imager below) after several hours of destaining.
IEF Marker:
Using the Gel Doc EZ Imager
1. Log into the computer using your Trinity ID
2. Open Image Lab
3. Load your gel onto the WHITE board, which is located in the drawer beneath the computer.
a. Be sure to wet the white portion of the board with a little water before placing the gel
on top
b. Make sure the gel is lying flat, without any air bubbles or folds. Use water to help you
manipulate the gel.
4. In the Image Lab window, select New Protocol
5. Under Gel Image, select Protein Gels- Coomassie Blue
a. Leave the defaults selected
6. Push Run Protocol
7. After the gel has imaged, if you want to adjust the contrast or the background, push the Image
Transform button (the one that is a half white and half black circle)
8. To save the file as a .tif, go to File-Export-Export for publication
a. Keep each of the default settings
b. Save to your Y drive as a .tif file
9. If you want to be able to manipulate the figure more, save the session as well. Go to File-Save
as and save it as a .scn file.
Bioinformatics Analysis:
1. For each protein, find the sequence for the protein from the given source. This is found at
National Center for Biotechnology Information center, which houses resources and Pubmed
(http://www.ncbi.nlm.nih.gov). On the website, you will see a pulldown menu of “Databases”
next to the search query. Pulldown the menu and select “Protein”
2. Type in the protein for which you are looking. There will be lots of entries so identify one that is
your protein and from your source.
3. Copy and Paste your Sequence into a Word Document. Also, copy and paste the “Accession #”
4. In order to determine the isoelectric point and the molecular weight, you will copy and paste
your sequence into ProtParam, a tool on the Expasy website, that will provide you with
theoretical chemical and physical properties for your proteins
(http://web.expasy.org/protparam/). Record the length of protein, pI and the MW.
5. For each protein sample from your gel, match the identity of the protein from the list.
Post-lab Analysis:
1. Identify each sample as one of the proteins from the list. Justify your answer based on the
bioinformatics and IEF analysis.
2. Discuss any difficulties in the identification and propose a reason for what the differences might
be.
3. Propose two alternative techniques to help with identifying the protein samples. Be sure to
discuss what information you would gain from these techniques.