Western Blotting University

Chapter 2: Electrophoresis and Transfer

Gary Ross, Ph.D.

Field Applications Specialist

Bio-Rad Laboratories
Western Blotting University

Chapter 2: Electrophoresis and Transfer

Section 1: Electrophoresis

Gary Ross, Ph.D.

Field Applications Specialist

Bio-Rad Laboratories
 Electrophoresis: Learning Objectives

1. Understand the principles of electrophoresis.

2. Identify the variables that affect electrophoresis.

3. Know how to select a gel for your application.

4. Know how to verify successful electrophoresis.

3
 Electrophoresis: Learning Objectives

1. Understand the principles of electrophoresis.

2. Identify the variables that affect electrophoresis.

3. Know how to select a gel for your application.

4. Know how to verify successful electrophoresis.

4
 Electrophoresis: Learning Objectives

1. Understand the principles of electrophoresis.

2. Identify the variables that affect electrophoresis.

3. Know how to select a gel for your application.

4. Know how to verify successful electrophoresis.

5
 Electrophoresis: Learning Objectives

1. Understand the principles of electrophoresis.

2. Identify the variables that affect electrophoresis.

3. Know how to select a gel for your application.

4. Know how to verify successful electrophoresis.

6
 Electrophoresis: Basic Principles
• Electrophoresis is the movement of
- Cathode
charged particles through a fluid under
- the influence of a uniform electric field.

- +
• Our goal is to optimize the relevant
+ parameters to separate our protein of
- + interest so it can be identified according
- to its electrophoretic mobility.
+
+ Anode

7
 Electrophoresis: Basic Principles
Factors that affect protein electrophoresis
• Strength of the electric field (voltage).
• Buffer composition:
o pH
o Concentration
o Ionic strength
• Gel composition
• Protein size, shape, net charge and
intermolecular interactions (e.g. EMSA).

8
 Electrophoresis: SDS-PAGE History
Raymond and Weintraub
introduce polyacryamide as
a stable, flexible, transparent
medium for gel
electrophoresis

1959

Raymond, Weintraub (1959)

9
 Electrophoresis: SDS-PAGE History
Raymond and Weintraub
introduce polyacryamide as
a stable, flexible, transparent
medium for gel
electrophoresis

1959 1967

Shapiro, Vinuela and Maizel

show that protein migration, in
the presence of SDS, is
dependent on the MW of the
polypeptide chains.
Shapiro, Vinuela, et.al (1967)

10
 Electrophoresis: SDS-PAGE History
Raymond and Weintraub Weber and Osborn study
introduce polyacryamide as 40 proteins using SDS-PAGE.
a stable, flexible, transparent Show a linear plot
medium for gel of protein mobility vs Log MW.
electrophoresis

1959 1967 1969

Shapiro, Vinuela, et.al show

that protein migration, in
the presence of SDS, is
dependent on the MW of
the polypeptide chains.
Weber, Osborn (1969)

11
 Electrophoresis: SDS-PAGE History
Raymond and Weintraub Weber and Osborn study
introduce polyacryamide as 40 proteins using SDS-PAGE.
a stable, flexible, transparent Show a linear plot
medium for gel of protein mobility vs Log MW.
electrophoresis

1959 1967 1969 1970

Shapiro, Vinuela, et.al show Laemmli produces

that protein migration, in the standard SDS-PAGE
the presence of SDS, is format still widely used
dependent on the MW of today.
the polypeptide chains.
Laemmli (1970)

12
 Electrophoresis: Experimental Choices
Western blots can detect proteins in a variety of states (e.g. denatured,
native, protein-protein complexes, protein-nucleic acid complexes, etc.).

Experimental choices:
• Native vs. Denaturing conditions

13
 Electrophoresis: Experimental Choices
Western blots can detect proteins in a variety of states (e.g. denatured,
native, protein-protein complexes, protein-nucleic acid complexes, etc.).

Experimental choices:
• Native vs. Denaturing conditions
• Reducing agents (β-mercaptoethanol, DTT) for disulphide bond
reduction

14
 Electrophoresis: Experimental Choices
Western blots can detect proteins in a variety of states (e.g. denatured,
native, protein-protein complexes, protein-nucleic acid complexes, etc.).

Experimental choices:
• Native vs. Denaturing conditions
• Reducing agents (β-mercaptoethanol, DTT) for disulphide bond
reduction
• Gel % (acrylamide/bis-acrylamide) that best resolves protein of
interest

15
 Electrophoresis: Experimental Choices
Western blots can detect proteins in a variety of states (e.g. denatured,
native, protein-protein complexes, protein-nucleic acid complexes, etc.).

Experimental choices:
• Native vs. Denaturing conditions
• Reducing agents (β-mercaptoethanol, DTT) for disulphide bond
reduction
• Gel % (acrylamide/bis-acrylamide) that best resolves protein of interest
• Selection of MW Standards for estimation of protein relative size

16
 Electrophoresis: Polyacrylamide Gels
• Pore size provides
unique sieving property to gel
matrix

• Acrylamide monomer and bis-

+ Ammonium persulfate and TEMED acrylamide (cross-linking) levels
are varied, producing uniform pore
sizes

• (%T) = total acrylamide gm/100ml

• (%C) = bis-acryl (gm)
bis-acryl + monomer-acryl (gm)

17
 Electrophoresis: Polyacrylamide Gels
• Typical polyacrylamide gel (%T) is anywhere from 4-20%.
- Low %T for high MW proteins
- High %T for low MW proteins

• Single percentage gels

- Best for resolution of proteins of similar MW.

• Gradient gels
- Best for screening broad range of MWs

• Lower percentage can help with downstream transfer

18
 Electrophoresis: SDS-PAGE
• Sodium Docecyl Sulfate (an anionic detergent) denatures protein structure

• SDS binds proportional to protein mass (1.4 g SDS/g protein),

approximately one SDS for every two amino acids.

19
 Electrophoresis: SDS-PAGE
• Protein movement depends on
- Electrophoretic mobility(µ), based on charge
- Gel sieving properties, based on size (MW)

20
 Electrophoresis: SDS-PAGE
• Protein movement depends on
- Electrophoretic mobility(µ), based on charge
- Gel sieving properties, based on size (MW)
• Binding of SDS imparts uniform
charge(-)/mass ratio* to proteins

21
 Electrophoresis: SDS-PAGE
• Protein movement depends on
- Electrophoretic mobility(µ), based on charge
- Gel sieving properties, based on size (MW)
• Binding of SDS imparts uniform
charge(-)/mass ratio* to proteins
• As a result, migration is dependent
mainly on the sieving property of the
gel; proportional to the (MW) of the protein.

22
 Electrophoresis: SDS-PAGE
• Protein movement depends on
- Electrophoretic mobility(µ), based on charge
- Gel sieving properties, based on MW
• Binding of SDS imparts uniform
charge(-)/mass ratio* to proteins
• As a result, migration is dependent
mainly on the sieving property of the
gel; proportional to the (MW) of the protein.

• *Some proteins bind different levels of SDS

causing them to migrate at a different rate than
expected (e.g. G-protein coupled receptors).

23
 Electrophoresis: Tris/Glycine/SDS PAGE
Laemmli gel format:

Tris-HCl gels
- stacking: Tris-HCl pH 6.8
- separation: Tris-HCl, pH 8.8

Running buffer: Tris-Glycine-SDS (pH 8.3)

Time

24
 Electrophoresis: Tris/Glycine/SDS PAGE
Laemmli gel format:

Tris-HCl gels
- stacking: Tris-HCl pH 6.8
- separation: Tris-HCl, pH 8.8

Running buffer: Tris-Glycine-SDS (pH 8.3)

Laemmli Sample Buffer :

(Tris-HCI pH 6.8, SDS, bromophenol blue,
glycerol, +/-BME) is still a commercial staple.

Time

25
 Electrophoresis: Tris/Glycine/SDS PAGE
Laemmli gel format:

Tris-HCl gels
- stacking: Tris-HCl pH 6.8
- separation: Tris-HCl, pH 8.8

Running buffer: Tris-Glycine-SDS (pH 8.3)

Sample Buffer : (Tris-HCI pH 6.8,

SDS, bromophenol blue, glycerol, +/-BME).

The discontinuous buffer

system provides stacking of the sample
Time volume for finer band resolution in the
separating gel.

26
 Electrophoresis: Additional Choices
• Pre-Cast vs Hand Cast gels
• Variable buffer options (not Tris-Glycine-SDS)
• Gel Staining options

27
 Electrophoresis: Pre-Cast vs Hand-Cast

Pre-Cast Gels Hand-Cast Gels

- Reproducible - Client design (%T, %C)

- Convenient, ready to use - More cost efficient
- Reliable gradient gels
- Saves time - Lower reproducibility
- Added time costs
- Added costs

28
 Electrophoresis: Tris-Acetate pH 7 Gels
• Need: Separation of large proteins (>200kDa)

• Solution: Tris-Acetate pH 7 gels

– Uses Tricine as trailing ion
– Optimal for SDS or Native PAGE applications

29
 Electrophoresis: Tris-Tricine gels

• Need: Separation of small proteins (< 5kDa)

• Solution: Tris-Tricine gels

– Uses Tricine as trailing ion
– Best for protein sequence analysis
• Tricine lowers Glycine background

30
 Electrophoresis: 2-D Gel Electrophoresis
• Need: Resolution of charge and Charge

Mass
size variants
• Solution: 2D gel platform

31
 Electrophoresis: 2-D Gel Electrophoresis
• Need: Resolution of charge and Charge

Mass
size variants
• Solution: 2D gel platform
– 1st dimension: Isoelectric focusing
separation of proteins; pH gradient
created by peptide ampholines.

32
 Electrophoresis: 2-D Gel Electrophoresis
• Need: Resolution of charge and Charge

Mass
size variants
• Solution: 2D gel platform
– 1st dimension: Isoelectric focusing
separation of proteins; pH gradient
created by peptide ampholines.

– 2nd dimension: IEF tube gel or IPG strip

equilibrated with SDS running buffer
and placed laterally on top of SDS gel.

• Best overall resolution of charge/size

variants within a protein sample.

33
 Electrophoresis: Visualizing Separation
• Stain-Free Gel
o UV-activated compound carboxylates
TRP residues, enabling visualization by red-shift
in TRP fluorescence.
o No staining or de-staining steps required.
o Sensitivity: 2-28ng
o Compatible with transfer and immunodetection.

• Coomassie Stain
o Coomassie dye binds to basic residues.
o Requires staining and de-staining steps.
o Bio-Safe Coomassie, Sensitivity: 8-29ng
o NOT compatible with transfer or
immunodetection.
34
 Electrophoresis: Visualizing Separation
• Silver Stain
o Fixation, staining and de-staining steps (2-3hrs).
o Sensitivity: 0.6-1.2 ng
o NOT compatible with transfer and
immunodetection.

• Oriole Fluorescent Stain

o Quick staining and de-staining steps (60-90min)
o Sensitivity: 0.5-1 ng
o NOT compatible with transfer or
immunodetection.

35
 Electrophoresis: Common Pitfalls

Vecteezy.com

Gel Overheating Choice of Gel Percent or Chemistry

Aggregates/Particles in Sample Sample Concentration too High/Low

Salts/Detergents Interfering Quality of Reagents

36
 Electrophoresis: Troubleshooting

Problem: Bands curved/smiling.

37
 Electrophoresis: Troubleshooting

Problem: Bands curved/smiling.

Solution: Gel was run too fast and overheated.

Reduce voltage.

38
 Electrophoresis: Troubleshooting

Problem: Misshaped bands

39
 Electrophoresis: Troubleshooting

Problem: Misshaped bands

Solution: Poor gel polymerization or running

buffer. Check gel polymerization, running buffer
and reagents.

40
Western Blotting University

Chapter 2: Electrophoresis and Transfer

Section 2: Protein Transfer

Gary Ross, Ph.D.

Field Applications Specialist

Bio-Rad Laboratories
 Protein Transfer: Learning Objectives

1. Understand the different methods of transfer.

2. Identify the variables that affect protein transfer.

3. Understand how to select a membrane for your

application.

4. Know how to verify successful transfer.

42
 Protein Transfer: Learning Objectives

1. Understand the different methods of transfer.

2. Identify the variables that affect protein transfer.

3. Understand how to select a membrane for your

application.

4. Know how to verify successful transfer.

43
 Protein Transfer: Learning Objectives

1. Understand the different methods of transfer.

2. Identify the variables that affect protein transfer.

3. Understand how to select a membrane for your

application.

4. Know how to verify successful transfer.

44
 Protein Transfer: Learning Objectives

1. Understand the different methods of transfer.

2. Identify the variables that affect protein transfer.

3. Understand how to select a membrane for your

application.

4. Know how to verify successful transfer.

45
 Protein Transfer: The Benefit of The Blot

• Small molecules (e.g. Coomassie) can diffuse through the gel matrix, but
macromolecules (e.g. antibodies) are too large.

• Transferring protein to a membrane makes immunodetection possible

by allowing antibodies to access protein on the membrane surface.

46
 Protein Transfer: Common Techniques

Microfiltration (dot blotting):

• Manual application of protein sample
onto membrane membrane

• Can apply small or large volumes

• 96-well format allows screening of
large number of samples
• No MW separation of proteins

47
 Protein Transfer: Common Techniques

Electrophoretic Transfer:
• Used for western blotting
• Applied voltage causes migration of
protein from gel to membrane
• Semi-dry or wet tank methods

48
 Electrophoretic Transfer: Semi-Dry Blotting

Semi-Dry Transfer:
• Transfer stack is in direct contact with
plate electrodes

49
 Electrophoretic Transfer: Semi-Dry Blotting

Semi-Dry Transfer:
• Transfer stack is in direct contact with
plate electrodes

• High rate of transfer, good for high

throughput

50
 Electrophoretic Transfer: Semi-Dry Blotting

Semi-Dry Transfer:
• Transfer stack is in direct contact with
plate electrodes
• High rate of transfer, good for high
throughput

• Uses minimal amount of transfer buffer

• Can overheat due to limited heat dissipation

51
 Electrophoretic Transfer: Tank Blotting

Wet Tank Transfer:

• Transfer stack is submerged in buffer
• Filter papers, gel and membrane need to be
placed between sponges and held in a plastic
cassette

52
 Electrophoretic Transfer: Tank Blotting

Wet Tank Transfer:

• Transfer stack is submerged in buffer
• Filter papers, gel and membrane need to be
placed between sponges and held in a plastic
cassette

• Slower rate of transfer

• Flexible voltage and time
• Reliable for high MW protein transfer

53
 Electrophoretic Transfer: Tank Blotting

Wet Tank Transfer:

• Transfer stack is submerged in buffer
• Filter papers, gel and membrane need to be
placed between sponges and held in a plastic
cassette
• Slower rate of transfer
• Flexible voltage and time
• Reliable for high MW protein transfer

• Uses large volume of transfer buffer

• Better cooling

54
 Membranes: Nitrocellulose

• Initial western blotting techniques used nitrocellulose

membrane

• Easily wetted by aqueous buffers for easy sandwich assembly

• Binding capacity: 80-100 ug/cm2

• Available in 0.45 µm and 0.22 µm pore sizes. Use small pore

size for small proteins (< 15 kDa)

• Works well for Far Red and near IR fluorescent signals (700-
800nm)

55
 Membranes: PVDF

• Polyvinylidene difluoride (PVDF) has a higher protein

binding capacity (150-200 µg/cm2) than nitrocellulose

• PVDF can provide strong chemiluminescent signal

• Must first be wetted using organic solvent (MeOH)

before making the transfer sandwich

• Also available in 0.45 µm, 0.22 µm and 0.1 µm pore sizes

• Proteins blotted onto PVDF can be directly micro-

sequenced (Matsudaira 1987)

56
 Membranes: LF-PVDF

• Low-fluorescence PVDF is advised for fluorescent blots

• Also performs well for chemiluminescent detection
• LF-PVDF is recommended for Stain Free applications
• Low auto-fluorescence/light scatter for low background
• Regular PVDF has high fluorescent background

57
 Protein Transfer: Confirming Success
• Use of Pre-stained MW standards:
– Historically, most popular technique
– Confirms efficient transfer of that selected lane/area only

• Total protein staining:

– Fluorescent, colorimetric, Stain-Free image of blot
– Varying sensitivities and backgrounds

58
 Protein Detection: Sypro Ruby Stain

• UV Fluorescence Detection
• Sensitivity: 2-8 ng
• MS compatible
• Doesn't block antigenic
epitopes

59
 Protein Detection: Ponceau S Stain
• Colorimetric Detection
• Sensitivity 100-200 ng
• Staining is reversible, wash
membrane prior to
downstream steps

60
 Protein Detection: Stain-Free Blot Imaging
• No staining / destaining steps
• UV Fluorescence Detection
– Tryptophan (Em) red shift
• Sensitivity 2 - 28ng
• Low background on LF-PVDF
and nitrocellulose papers
• Ideal for Total Protein
Normalization of immunoblot

61
 Transfer: Common Pitfalls

Vecteezy.com

Incomplete Transfer Low MW Transfer

Overheating Difficulty with High MW Proteins

Uneven Transfer Lack of Verification

62
 Protein Transfer: Troubleshooting

Problem: Swirls/bubbles

63
 Protein Transfer: Troubleshooting

Problem: Swirls/bubbles

Solution: Air bubbles or buffer was trapped

between the gel and the membrane during
transfer, causing local distortions. Carefully use
roller to remove any gaps or bubbles in
transfer stack.

64
 Protein Transfer: Troubleshooting

Problem: No bands/weak bands

65
 Protein Transfer: Troubleshooting

Problem: No bands/weak bands

Solution: Over-transfer or under-transfer.

Adjust transfer conditions, membrane type or
membrane pore size. Use total protein stain to
verify transfer.

66
 Electrophoresis and Transfer: Q & A

Questions?

67