Lab 7: Protein Electrophoresis (SDS-PAGE)
1 Introduction
Protein electrophoresis is an extremely popular technique in molecular biology. Simply, proteins (typically from a cell or tissue lysate) in an SDS-containing buffer are added to the top of a polyacrylamide gel. The SDS, a powerful anionic surfactant, serves to surround the protein, overwhelming its inherent charge. The protein surrounded by the negatively-charged SDS has a net negative charge approximately proportional to its mass. When a potential difference is applied across the gel, the negatively charged proteins migrate through it. Smaller proteins migrate more quickly through the gel, and the proteins are separated by size into ‘bands’.
Sodium Dodecyl Sulfide – PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is commonly followed by either total protein staining or transfer and Western blotting.
2 Objectives
• To separate proteins in a lysate by molecular weight
• To prepare a gel for Coomassie staining
• To prepare a gel for transfer and Western blotting
• Protean 3 manual
• Mini-transblot manual
• SDS-PAGE simulation
• The SDS-PAGE Hall of Shame
• Early Days of Gel Electrophoresis
• Image J

4 Reagents, Supplies, and Equipment
Note: Acrylamide/Bis-acrylamide is a neurotoxin!
4.1 Reagents
1. 0.5 M Tris-HCl, pH 6.8
This buffer is used when making SDS-PAGE sample buffer, and also when making the stacking gel.
2. 1.5 M Tris-HCl , pH 8.8
This buffer is used to make the resolving gel.
3. 10% SDS (5 g of SDS with dH
2
O, final volume 50 mL)
4. 0.1% SDS (dilute 10% SDS 1:100 with dH
2
O)
5. Resolving gel materials (amounts for 2-3 6% gels)
The resolving gel is the gel that is poured first; in it, proteins are resolved into discrete bands. dH
2
O 8.0 mL
Acrylamide/Bis-Acrylamide
1.5 M tris-HCl, pH 8.8
10% (w/v) SDS
10% (w/v) Ammonium persulfate (APS)
TEMED
The APS should be made fresh on the day of use.
μ
μ mL
150
10 μ L
L
6. Stacking gel materials (amounts given for 2-3 4% gels)
The stacking gel is poured on top of the resolving gel after it has finished gelling, with a ‘comb’. Protein samples are added to the individual wells formed by the stacking gel gelling around the comb. distilled H
2
O 3.0 mL
Acrylamide/Bis-Acrylamide
0.5 M tris-HCl, pH 6.8
1.25
10% (w/v) SDS
1% (w/v) Bromphenol blue
TEMED
50 μ L
5 μ L
10% (w/v) Ammonium persulfate (APS) 25
5 μ L
The APS should be made fresh on the day of use.
7. 5X gel-running buffer
This buffer is diluted to 1X with dH electrophoresis unit to operate.
2
O to make gel running buffer. Gel running buffer is used to fill the electrophoresis cell (or bath), keeping the gel wet and allowing the

(5x)125 mM Tris base 30.3 g Tris base
(5x)0.960 M glycine
(5x)0.1% SDS pH 8.3
144 g glycine
10
Add dH2O to solids until total volume is 1800 mL, bring to pH 8.3, then add more dH2O for final volume of 2000 mL. Store at room temperature.
8. 2X SDS-PAGE sample buffer
This is the buffer which protein samples are diluted before being loaded into the gel for analysis.
9. Standard protein, calf serum sample, and Molecular Weight standard (provided by instructor) and protein samples from Lab 5.
10. Transfer buffer - Do NOT pH, make fresh on the day of use
The transfer buffer is used when transferring proteins from the gel that you poured to a nitrocellulose membrane (for Western blotting).
25 mM Tris base
0.2 M glycine
3.03 g Tris base
15
20% methanol glycine
200 mL
11. Tris Buffered Saline with Tween-20 (TBST)
TBST is used to wash the nitrocellulose membrane.
20 mM Tris-HCl
137 mM NaCl
3.14
8.0
0.1% Tween-20
pH Add 900 mL dH
2
O, pH to 7.5, bring to final volume of 1000 mL w/dH
2
O. Store at 4 ° C.
4.2 Supplies
1. 1.5, 15, and 50 mL centrifuge tubes
2. 10, 100, and 1000 μ L pipette tips
3. 5 and 10 mL pipettes
4. Nitrocellulose paper
4.3 Equipment
1. Electrophoresis Equipment (casting stand, electrophoresis stand and apparatus)
2. Transblot Equipment (sandwiches, sponges)
3. Electrophoresis power supply

5 Protocol
You and your partner will produce and run two identical gels – one for Coomassie staining, and one for Western blotting.
5.1 Assembling the gel casting unit
1. Cover your lab bench with paper if you have not already done so.
2. Use distilled water to clean all electrophoresis equipment. Wipe with Kimwipe, and set the components out to dry. casting protean3.pdf
).
4. Check assembly for leaks using distilled water. Fill the space between the glass plates; if the unit leaks, reassemble and try again. If not, pour the water out and dry between plates using a folded paper towel, Kimwipe, or filter paper.
5. Begin water boiling for later use.
5.2 Pouring the Resolving Gel

1. Mix all resolving gel components together except the 10% APS and TEMED in a 50 mL centrifuge tube. Seal tube and tip back and forth gently to mix.
2. Add 10% APS and TEMED, seal tube and tip back and forth gently to mix.
3. Transfer solution to each of the two plate sandwiches using a 5 or 10 mL pipette. Do not fill all the way! There should be ~1 cm of space between the top of the short plate and the resolving gel level (right in the middle of the green plastic bar behind the glass plates).
It is important that you add the correct amount of gel. Too little and your resolution will be poor; too much, and there won’t be room for a stacking gel on top. Be careful to avoid bubbles, which will inhibit polymerization and distort protein migration.
Replace leftover gel in centrifuge tube.
4. Using a pipette or wash bottle, slowly and very gently add just enough 0.1% SDS solution to cover the resolving gel without disturbing it. The SDS solution is there to keep the gel from drying out and to protect it from oxygen, which will inhibit the reaction.
5. Wait 15-30 minutes. Check to see that the leftover gel in the centrifuge tube has polymerized, and if it has, tilt the gel former slowly to confirm that the resolving gel under the SDS solution has polymerized completely.
6. Pour the SDS solution into a Kimwipe, and rinse the top of the gel very gently with dH
2
O.
5.3 Pouring the Stacking Gel
1. Mix all stacking gel components together except the 10% APS and TEMED in a 15 mL centrifuge tube.
2. Add 10% APS and TEMED, seal tube and invert several times to mix. Add stacking gel solution to the top of the resolving gel using 5 mL pipette, returning extra solution to centrifuge tube. The stacking gel solution should almost fill the remaining space – leave ~ 2-3 mm between the top of the stacking gel and the top of the short plate.
Immediately thereafter, insert the comb. Start from one side and 'brush' air bubbles off to one side.
3. Wait 5-15 minutes. Check to see that leftover gel in the centrifuge tube has polymerized.
5.4 Assembling the electrophoresis unit
1. After polymerization, remove your gel sandwiches GENTLY from the gel casting apparatus and transfer them both into the electrophoresis unit as shown below ( from protean3.pdf
). When assembling the electrophoresis unit, the short plates must face inward !

2. Place the electrophoresis unit into the electrophoresis bath. Fill the inner chamber
(the small well between the two gels) with gel running buffer, and check for leaks.
3. Add approximately 250 mL of gel running buffer to the outer chamber (the clear plastic electrophoresis bath). 250 mL is about 4-5 cm of buffer, measured from the bottom. While the inner chamber must be filled, the outer chamber need not be completely filled.
4. Remove comb carefully and gently rinse each loading section of the gel gently with the gel running buffer using a 1000 μ L pipette.
5. Set up equipment near power source.
5.5 Electrophoresis
Before beginning electrophoresis, you will need to know the concentration of your protein samples from Lab 5, which you calculated during Lab 6.

1. Add 1 part 2X sample buffer to 1 part protein samples from Lab 5 in separate tubes.
For this experiment, 50 μ L of 2X sample buffer added to 50 μ L of each protein sample should be more than sufficient.
30 L of each standard (be sure to choose the standard appropriate to your sample, collagen I or collagen II) to 30 μ L sample buffer in separate tubes.
30 L of the calf serum sample to 30 μ L sample buffer in another tube.
4. Heat protein samples and standards ( not the molecular weight ladder) at 100 C for 5 minutes. Make sure that your caps are securely fastened. If available, use plastic cap protectors to make sure that the caps don’t pop off during boiling.
5. Put your samples on ice for ~60 seconds.
6. Centrifuge samples briefly to remove bubble and pellet any undissolved cell extract.
Your protein will be in the supernatant; any pellet should be left undisturbed.
7. Using a 100 μ L pipette, add protein samples to the wells in the gels as shown below.
Be very careful!
It’s easy to miss a well entirely if you’re not paying attention. Pipette very slowly. You will be loading two identical gels.
Your standard protein will be either collagen I or collagen II. Ask the instructor which you should use.
Lane 1: 40 μ g calf serum (20 μ L = 10 μ L standard + 10 μ L 2X sample buffer)
Lane 2: 2D sample protein, 3T3
Lane 3: 3D sample protein, 3T3
Lane 4: 2D sample protein, Rex
Lane 5: 3D sample protein, Rex
Lane 6: 0.1 μ g standard protein (20 μ L = 10 μ L standard + 10 μ L 2X sample buffer)
Lane 7: 0.5 μ g standard protein (20 μ L = 10 μ L standard + 10 μ L 2X sample buffer)
Lane 8: 1.0 μ g standard protein (20 μ L = 10 μ L standard + 10 μ L 2X sample buffer)
Lane 9: Molecular weight ladder (8 μ L, no 2X sample buffer added, no heat )
Lane 10: empty
The gel can be overloaded two ways - too much protein, or too much volume. Too much protein will separate badly. The maximum volume depends on the type of comb and the spacer plates used. or an 8-well comb using 0.75 mm spacer plates, I suggest using no more than 25 μ L sample volume.
Your goal is to load no more than 40 μ g of sample protein per well in no more than a
25 μ L volume . If you can’t load 40 μ g of protein, load as much as you can without going over the 25 μ L limit.
8. Run the gels at a constant 150 V until the blue dye front has nearly run out of the gel
(approximately 60 minutes). For a large protein like collagen, it’s important to run the gel for a relatively long time – otherwise, the collagen will not enter far enough into the gel to separate the collagen band from the other high molecular weight bands
(the resolution will be poor).
5.6 Disassembly and Transfer
1. When finished, disassemble the electrophoresis unit.

2. Prepare a bath of transfer buffer and a bath of distilled water.
3. Use a wedge to gently separate one of the glass plates from the gels. The gel might tend to stick at the top, where the stacking gel was. It doesn't matter which plate you remove, as long as you can remove one leaving the gel on the other. Be careful! It's easy to tear the gel at this point.
4. Carefully take a folded Kimwipe and place it atop the stacking gel (small gel on top with comb) without touching the resolving gel (larger gel on bottom without comb).
The edge of the towel should line up with the separation between resolving and stacking gel. Press the paper towel firmly against the stacking gel, then pull the paper away. The stacking gel should neatly come with the paper, and can be discarded. If it doesn't all come off at one, try again.
5. Submerge the gel in the transfer buffer bath. If necessary, use the wedge to loose the gel into the buffer bath.
6. During those 15 minutes, use a wedge on the other gel to separate the glass plates.
7. Use a paper towel to remove the stacking gel from this gel, as before.
8. Submerge the gel you’ve just removed the stacking gel from in the bath of distilled water and separate it from the glass plate. Store in the refrigerator.
9. Also during those 15 minutes, cut a piece of nitrocellulose membrane and pieces of filter paper so that their size matches that of the gel. Always try to avoid touching the nitrocellulose membrane, except at the edges.
Incubate this membrane in transfer buffer for 5 minutes (after the gel has incubated for 10 minutes so that both will be ready simultaneously).
10. Dip the filter paper and sponges into the transfer buffer. Using the mini trans-blot unit
( transblot.pdf
) and the gel that’s been incubating in transfer buffer, make a sandwich of the following:
11. Before sealing it, roll a pipette over the top paper layer of the sandwich to ensure that the membrane and the gel are pressed against each other without any air bubbles that will interfere with the transfer.
12. Insert this sandwich into the blotting apparatus ( transblot.pdf
, pg 6) with another group’s sandwich, then fill the transblot apparatus with transfer buffer.
13. Run at 25 V overnight in the refrigerator to allow transfer.
14. Tomorrow morning, your instructor will turn off mini trans-blot unit, remove the mini trans-blot sandwich, and carefully peel the nitrocellulose membrane from the gel. The membrane will be washed with and stored in TBST until next week.

6 Discussion
• How might we change this protocol if we were interested in a different protein?
7 Homework
Epidermal Growth Factor (EGF) is a fairly important regulatory hormone. Almost all cells have at least one form of the epidermal growth factor receptor (EGF-r). EGF binds to the
EGF receptor on the cell surface, and the intracellular portion of the EGF receptor acts as a protein kinase, whose enzymatic activity is regulated by whether EGF is bound or not.
Basically, when EGF and EGF-r are bound, the receptor is ‘on’ and activates proteins inside the cell. When it’s just EGF-r by itself, it’s ‘off’, and has very little activity.
Like many cell surface receptors, when activated, EGF-r is actively internalized. When it constantly receives an EGF signal, the cell reduces the amount of receptor on it’s surface
– just as your eyes adjust to a strong signal (bright light) by attenuating how sensitive the eye is to light (by adjusting the pupils).
Below is a Western blot of EGF receptor for two cell types: 184A1 HMEC and B82 fibroblasts (much like your 3T3 fibroblasts in many respects). The ‘time in EGF’ is how long the cells were exposed to EGF before they were lysed and harvested. The longer the cells are in EGF, the fewer receptors they have.
Using NIH image, calculate what percentage of EGF receptors remain after 3, 6, 8, and
24 hours of EGF treatment.
Consider that 100% is the amount of receptors for cells which have received no EGF treatment (the 0 hour band). You’ll want to compare the darkness of the ‘0’ band with the other four bands for each cell type.
This image is posted on the web page as a TIFF file for NIH image called ‘wiley.tif’ (gel courtesy of Dr. Steven Wiley, U. Pitt).