Gel electrophoresis and

staining procedures
BIOC23 Week 6 Lecture
Modified from Brunt 2022

1
 What is gel electrophoresis?
• process by which charged molecules are separated within a matrix in
an electric field based on their differential migration (mobility)

• Different size formats and

can be vertical or horizontal

2
Gel Electrophoresis

• The rate of migration of the molecules depends on:

• strength of the electric field
• net charge of the molecule
• size and shape of the molecule
• ionic strength, viscosity and temperature of the medium in
which the molecules are moving.

3
Principle of Electrophoresis
Electric field strength (volts)

Charge on molecule
velocity
Distance b/w electrodes
Frictional coefficient
(function of molecule radius
and medium viscosity)

Can increase velocity by increasing E (electric field) or

decreasing d (distance between electrodes)
But remember:
Temp will also increase and could melt gel!
4
Solid matrices for gel electrophoresis
• Solid matrices prevent loss of resolution by diffusion and to maintain the separation after
electrophoresis is complete.
• Must be strong, hydrophilic (to prevent hydrophobic interactions between the medium and
protein), uncharged and stable over a wide range of temperature, pH and osmolarity
• Must be able to control and adjust the porosity (pore size is important since it is a major
contributor to the frictional coefficient)
• The sieving effect of the support medium and the charge to mass ratio of the molecules
fractionates the molecules in the sample
• paper and starch were the first support media
• Today: Polymerized acrylamide (note: neurotoxin in unpolymerized form) for protein
separation and some nucleic acid work and solidified agarose for much of the RNA/DNA
separations
• The molecular sieving property of the semi-rigid gel helps to separate large ionic compounds
such as proteins.  5
Migration of a molecule
Mobility is defined as the rate of migration traveled with a voltage gradient
of 1V/cm

 it is measured in square centimeters/second/volt.

 can determine relative mobility of proteins (Rf) as the ratio of the

distance each protein migrates to that of a small amount of anionic
dye (tracking dye: Bromophenol blue or xylene cylanol) or

 the high charge to mass ratio of the dye causes it to migrate near the
electrophoretic front and ahead of proteins
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Separation of proteins
• proteins have a net charge at any pH other than their isoelectric
focusing point (pI)
• Therefore, proteins will migrate when placed in an electric field
• for most proteins the pI is in the range of 3 to 10 with the majority
having a pI of < 8.
• therefore at a pH of 8 or higher a majority of proteins have a net
negative charge and will migrate toward the anode (positive
pole)

A buffer is required to keep pH constant:

Electrolysis of water generates H+ at the anode and OH– at the cathode
7
 Polyacrylamide gel electrophoresis (PAGE)

• Polyacrylamide gels are formed by the copolymerization of

acrylamide (CH2=CH-CO-NH2), which is a water soluble monomer with
a cross-linking agent called N,N’-methylene bisacrylamide (CH2=CH-
CO-NH-CH2-NH-CO-CH=CH2) also called bis
• forms a three-dimensional lattice
• Polymerization is typically accomplished using a chemical reaction.
• The most common chemical method is by using ammonium persulfate (initiator)
and the quaternary amine, N, N, N', N' – tetramethylethylenediamine (TEMED)
(catalyst)

8
 TEMED reacts with these
free radicals to produce TEMED
free radicals which reacts
with acrylamide to induce
polymerization

BIS crosslinks the adjacent acrylamide chains

9
 Lattice of co-polymerization
The average size of the
pores in a polyacrylamide
gel can be controlled by
varying the amount of
monomer used or
amount of cross-linking
(*higher % acrylamide =
smaller pores)

• For a given gel composition, the pores have a statistical distribution of pore sizes
• Relatively small proteins migrate in the gel with minimal resistance
• Larger proteins will be slowed because they cannot pass through all the pores in the gel
• Very large proteins may not enter the gel at all
• Can use a single percentage gel or a gradient gel, which gives a wider range of
separation/resolution 10
SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel
Electrophoresis)
• Remember that proteins (in contrast to DNA) do not have a fixed charge-to-mass ratio (in DNA,
there is one negative charge per nucleotide).
• This is why, in their native form, proteins cannot be separated by size using non-denaturing
electrophoresis (but DNA can).
• SDS, artificially creates for proteins, the situation that exists naturally for mixtures of DNA
fragments: a fixed charge to mass ratio
• sieving effect is the principal means of fractionation
• Separation is based on size, since all proteins will have identical charge to mass ratios due to
coating with negatively charged SDS
• Note that the sieving effect in gel electrophoresis is different from that in gel-filtration: i.e.
molecules are not excluded since it is a mesh-like substance rather than a bead.
• larger molecules travel more slowly through the sieve than small molecules
• SDS sample buffer (Laemmli’s buffer). Contains SDS, Tris buffer pH 6.8, and often a reducing agent
(BME or DTT) can be added. Heat (70-100C is required) 11
SDS-PAGE: Protein population has the same charge to mass ratio so that separation is
based on molecular weight with charge having little to no influence
• Denature proteins with the
detergent sodium dodecyl sulfate
(SDS, an anionic detergent)
• Causes the folded proteins to
become denatured into extended
“rods” coated with SDS
• Average of one dodecyl sulfate
molecule for every two amino acids
• Each SDS molecule has two negative
charges at pH values used for
electrophoresis (pH 8-9), so proteins
will all be negative with essentially
an identical charge to mass ratio
mixtures of proteins will
Band appearance upon
-mercaptoethanol migrate through SDS-PAGE
staining
(reducing agent reduces disulphide based on their relative
bonds between cysteines) molecular weights.
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• Loading buffer (GSB) which is added to the protein samples
(before or after denaturation), it can be added to the SDS
sample buffer) contains:
• SDS (to denature and “charge” protein)
• -mercaptoethanol (reduces disulfides)
• Buffer (neutral pH, pH 6.8)
• Tracking dye (bromophenol blue) blue at alkaline conditions
• Glycerol (density)
This is how we monitor progress of
electrophoresis
(i.e. dye marker Does NOT bind to
protein)

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separation in the resolving portion of the gel:
upper reservoir

lower reservoir

Stryer 2002

14
What acrylamide % is best for your protein of interest?

15
• Continuous Buffer System (can be used with or without SDS, but today used usually
without: native separation)
• In continuous buffer systems, the composition of the gel buffer and running buffer are
essentially identical throughout the gel.
• consist of a single separating gel and use the same buffer ions at the same pH throughout
the sample, gel, and electrode reservoirs.

• Discontinuous Buffer Systems (used with SDS usually)

• The discontinuous buffer systems employ different buffer ions and pH in the gel and in the
electrode reservoirs.
• Samples are loaded onto a non-restrictive large pore gel, called a stacking gel, which
overlays a smaller pore resolving gel.
• The major advantages of discontinuous buffer systems are that relatively large volumes of
dilute protein samples can be applied to the gel
• resolution is much greater than that obtained with continuous systems.
• This increased resolution is a direct result of the way proteins concentrate, or stack, into
narrow zones during migration through the large-pore stacking gel, and destack or
resolve, under the given conditions in the small pore resolving gel. 16
Schematic of SDS discontinuous PAGE
(Laemmli (Nature 227:680-685 [1970]) and modified by Studier for slab gels): denaturing system

samples: tris pH 6.8, SDS, BME and tracking dye

• as electrophoresis continues protein
molecules reaching the resolving gel wells: formed by teeth or comb:
will become greatly retarded, Cathode - can vary size
allowing trailing proteins to catch up
Stacking gel: low percent acrylamide usually
• this ensures that the volume of 0.12M tris pH 6.8, 0.1% SDS 5-5.5% (no sieving)
sample presented to the
separation gel will be much •sieving: i.e. separation
Resolving gel based on size:
smaller than the volume
•usually 7.5% to 15%
1.5 M tris pH 8.8, 0.1% SDS
initially loaded onto the or gradient gel
stacking gel.
-each section poured
separately and
Anode +
allowed to polymerize
running buffer: 0.12M tris pH
8.3,1.9M glycine, 0.1% SDS
(glycine
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 TRIS-Glycine system
http://www.youtube.com/watch?v=koe0Xv7a2MM&feature=related
http://www.youtube.com/watch?v=pV_HdqKOTSo&NR=1
• Glycine from the upper reservoir (at pH 8.3) enters the low pH of the
stacking/sample (pH 6.8) predominantly in a neutral zwitterionic form with only a
small fraction (1%) in the negative glycinate form
• The Cl- ions of the Tris buffer remain the effective current carriers at pH 6.8 and
migrate rapidly towards the anode.
• During the electrophoretic separation in the stacking gel the Cl- ions become
depleted at the cathode end of the gel (top),forming an increasing concentration
gradient towards the anode (bottom)
• The SDS-coated proteins and the dye have charge to mass ratios greater than
that of glycine but less than that of Cl- and end up stacked in a thin band
sandwiched between the Cl- and the glycine ions at the interface of the stacking
and separation gel
• when the stacked bands enter the higher pH of the separation (resolving) gel
glycine becomes anionic, carrying a higher charge to mass ratio than the proteins
• the newly formed glycinate ions move faster than the proteins joining Cl - at the
front
• a new concentration gradient is formed, in which the proteins and the tracking
dye carry the current in the trail of the Cl- and the glycinate ions
• proteins separated by the sieving effect of the separation gel according to their
relative molecular weights
• dye has a higher mobility than the proteins and reaches the bottom of the gel first
so electrophoresis is terminated before proteins can run off the gel
• keeping the SDS concentration at 0.1% (w/v) throughout the gel ensures the
protein is always coated with SDS
 Stain with coomassie blue to
visualize proteins on the gel

• Coomassie brilliant blue R-250 is closely related to the G-250 used in the Bradford protein
quantification method (G-250 can also be used to stain)
• Protein is stained and fixed at the same time (with dilute acetic acid (5%) and
methanol (40%) (30 min)
• Based primarily on interactions with arginine and weak interactions with
tryptophan, tyrosine, phenylalanine histidine and lysine
• Destained with acetic acid and methanol (must remove all background) (newer version can
destain with water)
• Can then be photographed and or scanned for quantification
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 Staining techniques
•
Zinc and copper staining are negative staining techniques in which the background of the
gel is stained and the proteins are visualized as clear spots. Zinc and copper does not stain
SDS, which is coating the resolved proteins, but does stain the polyacrylamide gel matrix
(not routinely used).
• Coomassie Blue staining (generally .3 to 1 ug per band) is a quick, easy and cheap and
multiple protocols and pre-made reagents are available.
• Silver staining (2 to 5 ng per band) can give excellent results but is a time consuming and
expensive technique that can interfere with MS analysis if not done properly. The
advantage of silver staining is its sensitivity, although the method lacks specificity and
dynamic range, generally negating quantification of proteins in a gel.
• Fluorescent stains are easy and relatively quick to use but some, like SYPRO ruby other
trademarks are more expensive. These dyes are regarded as both selective and
quantitative allowing the intensity of protein spots to be directly correlated to the
quantity of protein present. These stains are as sensitive as Silver staining and require
more expensive equipment for visualization.
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Example: Compare fractions:
proteins are stained with
coomassie blue

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• Polyacrylamide has a limited capacity for protein addition (loading).
• Overloading results in precipitation and aggregation of proteins, producing streaks and
smears.
• Underloading results in complete disappointment, as one may detect only the most
abundant of polypeptides, if that.
• The objectives of sample preparation is to put the proteins into a denaturing buffer, making
them suitable for electrophoresis, and to adjust the concentrations of sample so that an
appropriate amount of protein can be loaded onto a gel. 22
You usually determine molecular weight of an unknown protein (or other
macromolecule) using the distance migrated of a known (ignore the dye front).
Migration is inversely related to the log MW (Mr)

- Use a series of standards to carry

this out
- Not linear across the large range of protein
sizes
- May see more than one slope for
a large range of proteins on one gel

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Other types of gel electrophoresis
• triton/ acid urea gels: basic proteins, slab gels
• nondenaturing (native) gel electrophoresis (detergent is not used):
• format slab or tube
• similar buffer to SDS PAGE but no detergent
• separates based on net charge and size
• stain for enzyme activity
• structure studies
• capillary electrophoresis: high speed/high performance/ high resolution
(1000’s columns): used in large scale nucleotide sequencing (separated in
a capillary tube and separation is observed via a chromatogram)
• agarose gel electrophoresis: both nondenaturing (DNA) and denaturing
(RNA), more elaborate PFGE (Pulse field) gel system 24
• isoelectric focusing (IEFs)

• migration of proteins is due to their charge i.e. migrate to their approximate pI

(remember post-translational modifications along with amino acid composition
will effect charge)
• historically used for analytical purposes performed in tube gels (although flat
bed is more common now with fixed pH gradients)
• now preparative, isolation of large amount of protein for analysis
• a pH gradient is set up within the IEF gel by the addition of polymeric buffer
components called ampholytes.

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Isoelectric focusing (IEFs) gels
• migration of proteins is due to their charge (remember post-translational modifications
along with amino acid composition will affect charge)
• the acrylamide concentration is low (5.5%) to prevent separation by size
• pre-running sets up the pH gradient in the case of non-immobilized pH gradients by:
• each ampholytes (buffer component) migrating to a position where the pH equals its pI
• alternatively can use gels (flat bed) that has immobilized pH gradient covalently attached to an
acrylamide gel).
• samples resuspended in urea (to denature) and ampholytes are introduced and the
proteins move in the current to a position in the gel where the pH is equal to the pI
• Can be used to detect small changes in proteins and high resolution
• High cost due to ampholytes, special equipment, more technically difficult, must run
identically every time (volt/hours)
• Results in proteins focusing in a narrow band
• Historically used for analytical purposes performed in tube gels; Now preparative, isolation of large
26
IEF-two dimensional gel pI in the first dimension
electrophoresis: combines
IEF in the first dimension
and SDS-PAGE in the
second dimension either using a
Fixed pH gradient
workhorse of proteomics: or a gradient set
can see up to 1000 protein up by current
“spots” or more

Size in the second

- Used to identify proteins dimension

which change in expression

-Can then analyze (Mass
spec)

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28
 29
http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm
Example of what you might see if using multiple
purification techniques to purify a protein while
monitoring the process for protein amount,
specific activity, and gel electrophoresis

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 LAB4 Protocol Day 3: Electrophoresis of Serum Proteins (**Complete Table 2 and 3 before lab!**)
Prepare Samples of Fractions B, C, D based on determination of protein concentration from Bradford assay

}
1.75µg Fraction B = ____µL of B + ____1x SDS sample buffer
7.5µg Fraction C = ___µL of C + ____ 1x SDS sample buffer Add GSB
7.5µg Fraction D = ___µL of D + ____ 1x SDS sample buffer to 20%

Place samples B, C, D, E, F in a heating block (with cap closure) at 100°C for 5 min.
Remove from heat and cool for 5 min. Centrifuge 5-10 seconds. Load 24µL into assigned lanes.

M RS B C D E F
8-16% gel M= MW markers
RS = total rabbit serum

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Invitrogen Benchmark™ Protein Ladder
LAB4 Protocol Day 3: Electrophoresis of Serum Proteins
After running gels at 125 volts for approximately 40 mins, you will remove the gel
and stain it with Coomassie Blue.
Why is this necessary?

https://i.ytimg.com/vi/ViJYNOSm6Ww/maxresdefault.jpg
https://assets.thermofisher.com/TFS-Assets/BID/product-images/EI0002_650x600.jpg-650.jpg

https://i.ytimg.com/vi/-H8GU_90Gak/hqdefault.jpg

You will receive a file with your results: the following lab your will present a short oral
presentation of your gel results 32
Presenting your gel IgG data in lab (week 7)
• After lab this week you will receive an image of your stained gel from
LAB4
• BEFORE your next lab (week of Feb 27-Mar 3), each group will
prepare a figure of your gel with a title/figure legend (look at article for
examples)
• Prepare a one minute summary of the data, which you will present in
lab (there will be a projector)
• This is participation-based grading meant to help guide discussion
about the data and about preparing figures/figure legends

33
IgG Report (Due March 12)
Tips for methods section:
• Look at examples. They are MUCH more brief than protocols
• Subheadings are used for separate types of protocols (there is some flexibility as
to what those subheadings are – you choose how you want to group/present information)
• Paragraph/full sentence format. (past tense/passive voice commonly used: “Samples
were processed…”)
• Volumes are often NOT written out (but concentrations/buffer
composition/key reagents are)
• Don’t need to describe common lab manipulations. (ex. how to pipette, or
how to make the wester transfer “sandwich”)
• For writing your report, write for a target audience that has a general science
background

34
 Lab#5 - GST
What is the objective of today’s
experiment?

To purify a GST-hsp90 fusion

protein from an E. coli cell
extract by affinity
chromatography using a spin
column format. This purification
is based on the affinity that the
enzyme GST (glutathione-S-
transferase) has for its natural
substrate glutathione.
https://www.goldbio.com/documents/1046/Affinity%20GST%20Purification%20Protocol%20utilizing%20Glutathione%20Bulk%20Resin.pdf

35
Protocol Day 1: Procedure for GST fusion protein isolation

Plate on LB agar plus AMP

Pick a
single
colony

YTA media Grow O/N

37°C shaker

1.5mL uninduced cells (UI)

UI How do we test to see if our
2.5mL cells have induced well?
O/N
culture
Add 10µL of 100mM
IPTG per mL of culture;
Fresh YTA media; grow grow 2 hours
to A600 of 0.6-0.8 36
Induced cells (I)
 Protocol Day 1: Procedure for GST fusion protein isolation
WHY?
Discard
supernatant START
5000 rpm
HERE *
5 mins. RT
UI Add 150µL SDS
sample buffer
1.5mL
uninduced UIC
(UI) Sample Prep for
UI SDS-PAGE
(UI) Pellet
(Uninduced cell extract)
(section C)

1.5mL Add additional Repeat Discard

centrifugation supernatant
1.5mL
I

4mL 1.5mL pellet

induced induced (I) I I
(I) (I) pellet
I
37
 Protocol Day 1: Procedure for GST fusion protein isolation

10 µL 30 µL SDS sample buffer

160µL
1xPBS
+protease Prepare for SDS-PAGE
inhibitors IC gel#2 on day 3
I
Resuspend (Induced cells)
150µL 50°C water bath
Liquid Nitrogen
REPEAT
ADD:
3µL of 10mg/mL
lysozyme I
7µL 100mM DTT 10 times 50
2µL 1mg/mL DNaseI

What is the role of

each component? 38
Protocol Day 1: Procedure for GST fusion protein isolation
After Transfer
freeze/thaw supernatant
12,000 rpm Remove 7µL supernatant
cycles
10 mins. RT into new tube – label “E”

I
I I Add 13µL SDS
WEB sample buffer
This is your induced
Whole cell Extract (for
Dispose
Blot)
pellet.
WHY?

After taking 7µL aliquot

for sample WEB,
use the remainder for
I Glutathione affinity
chromatography
39
 Protocol Day 1: Procedure for Glutathione Affinity Chromatography
Place column in clean Spin 3000 rpm for 1 min. RT
microfuge tube
Resuspend Loosen cap ¼ turn
resin with
gentle
vortex

Snap off bottom

closure
https://assets.fishersci.com/TFS-Assets/LSG/product-images/89868-column.jpg-650.jpg
Discard buffer in microfuge tube; replace bottom cap.

Inversion 10 min @ RT
Spin 3000 rpm 1 min. to
collect flow through

~ 160µL
I
40
What do you expect to be in the Flow through? In the column?

Place column in clean

REPEAT
microfuge tube

Add
300µL
This will
1xPBS
Spin 3000 Spin 3000 remove
rpm 1 min. rpm 1 min. unbound
contaminants

Collect flow
through
(tube FB)
FB What is happening
during inversion?

Add 100µL Inversion 10 min @ RT

Glutathione Spin 3000
elution rpm 1 min. Collect eluted
buffer proteins (tube GB)
GB
41
Protocol Day 1: Sample preparation for SDS PAGE Need:

UIC IC WEB GB FB

(UI) (IND) induced Whole Eluted GST Flow-through

cell Extract
Remove 10µL
8µL to 7µL “WEB” 12µL
new tube and 13µL SDS

Heat 100°C 5 mins, then add GSB.

Add Add
12µL 10µL Gel#1 (for Blot)
GB-1 SDS FB-1
SDS
Add Add
Add WEB-1 GB-1 FB-1
8µL 4µL Heat 100°C 5 mins. Heat 100°C 5 mins.
2µL
GSB GSB
GSB UIC-1 IC WEB-1
Gel#2
Add Add
4.8µL FB-1 4µL
GSB GB-1 UIC-1 IC
GSB 42
Protocol Day 2: SDS PAGE and Western Transfer

Ponceau S
1 2 stain and
rinse with
H20

3
5 4
WEB-1,
GB-1, FB-1

Wash membrane in
PBST at intervals;
incubate with 1°Ab-HRP

43
Modified from: https://d3i71xaburhd42.cloudfront.net/aad5857cb24a7862d7a970c64611874a0fb6610b/2-Figure1-1.png
Protocol Day 2: SDS PAGE and Western Transfer

Gel #1: Heat samples WEB-1, GB-1 & FB-1 @ 100°C for 2 mins (previously stored at -20°C from last week)

Lane 1 2 3 4 5 6 7 8 9 10
M WEB GB FB + M WEB GB FB +
GEL#1: Two groups will load samples on one gel as indicated

1. Pre-stained molecular weight markers (“M” 5μL)

12% polyacrylamide gel 2. sample WEB-1 ( induced whole cell extract) load 20uL
3. sample GB-1 (eluted GST fusion protein) load 20µL sample
1X SDS tris/glycine running buffer 4. sample FB-1 (flowthrough proteins that did not bind
column) load 20µL
130V for 1.5h 5. + positive control (purified GST protein or fusion protein
provided) 0.5 μg

Load gel and run until dye front is near bottom of gel. Remove gel; trim off wells. Prepare for transfer.

44
Protocol Day 2: SDS PAGE and Western Transfer

• Why is it important that the gel be placed on the negative electrode

side of apparatus?

Set up transfer for

1hour at 300mA.

*
https://assets.thermofisher.com/TFS-Assets/BID/product-images/EI0002_650x600.jpg-650.jpg

Once transfer is complete, membrane is removed and stained with Ponceau S.

We stain and image the membrane before immunodetection. Why is this important? *
Next, we stain the gel (post transfer) with Coomassie Blue Stain. What information will this give?
45
Protocol Day 3: Immunoblot https://youtu.be/098qhQ2V_B4 First watch 4min video on immunoblotting

Block with 5% non-fat Place blot in 15mL 1:30,000

dry milk in PBST dilution of anti-GST/HRP conjugate
Wash stained (Tween 0.1%) in 1% BSA/PBST (Tween 0.1%)
membrane with
Rinse membrane
PBST (Tween 0.1%)
with 40mL PBST
(Tween 0.1%) 5min
REPEAT

Agitate for 45min

60 min
2 x 5min https://youtu.be/K-0fkJy2QCU https://youtu.be/58yCRC69dUk
Watch Video on detection phase Video: hrp conjugated and ECL

Place blot in 15mL of Develop blot

Rinse membrane Rinse membrane using CCD camera
with 40mL PBST ECL substrate for 5 min
with 40mL PBST
(Tween 0.3%) (Tween 0.1%)
15min 10min
REPEAT REPEAT

46
Protocol Day 3: SDS-PAGE Gel #2 - Uninduced vs. Induced Cells

Lane 1 2 3 4 5 6 7 8 9 10 GEL#2: Three groups will load samples on one gel as indicated
Pre Un UIC-1 IC UIC-1 IC UIC-1 IC Un
1. Pre = Pre-stained molecular weight markers (5μL)
2. Un = Unstained molecular weight markers
3. Sample UIC-1 (uninduced) load 10µL sample
4. Sample IC (induced) load 20µL
12% polyacrylamide gel

1X SDS tris/glycine running buffer

130V for 1.5h

Load gel and run until dye front is near bottom of gel.
Remove gel; Trim wells. Stain with Coomassie Blue.
47
Electrophoresis Learning objectives
• Predict how gel electrophoresis migration velocity of a protein will be affected by
protein size, protein charge, electric field strength, distance between electrodes,
and pore size (% acrylamide).
• Explain why SDS and reducing agents (i.e. beta-mercaptoethanol) are used during
analysis of proteins by gel electrophoresis. How would the addition/omission of
these impact results?
• Describe movement of proteins in terms of cathode and anode
• Explain how a stacking gel can sharpen protein bands
• Explain why it is important to select the correct gel % (based on your protein of
interest) in SDS-PAGE
• Describe what properties of proteins separation is based on 2D-IEF and discuss
when this approach would be useful 48