Week 8: Laboratory Exercise 8

Electroblotting from Polyacrylamide Gels

I. INTRODUCTION

Blotting is a technique for the electrophoretic transfer of DNA, RNA or protein to a suitable
membrane. The method most commonly used for the electrotransfer of proteins to nitrocellulose is
that reported by Towbin et al. (1979). This technique was patented in 1989 by William J. Littlehales
under the title "Electroblotting technique for transferring specimens from a polyacrylamide
electrophoresis or like gel onto a membrane." Transfer of the proteins can be carried out using
several methods such as vacuum, capillary or electric field. Electroblotting is by far the most wide-
spread technique which utilizes either vertical buffer tanks or semi-dry blotting.

In order to take advantage of this technique for the purpose of amino acid analysis or N-
terminal sequencing, the proteins must be transferred to a membrane that is stable to the chemicals
used in these analytical procedures. For protein sequencing and amino acid analysis the proteins
are transferred to a chemically stable membrane, polyvinylidene difluoride (PVDF). PVDF
membranes bind proteins primarily through hydrophobic interactions and are commonly used for
their chemical resistance as well as physical stability. Electrophoretic transfer is performed by
placing the gel next to the membrane in a special cassette that, in turn, is placed in a tank of
electrophoretic buffer (tank electroblotting).

Proteins are first separated by SDS-PAGE, the gel is removed from the electrophoresis
cassette and equilibrated in transfer buffer without methanol. The PVDF membrane is "activated"
by dipping it in methanol; it is then placed in transfer buffer containing methanol. The gel-PVDF
sandwich is placed in a specially designed holder that in turn is placed in the buffer-containing
electrophoresis unit. Upon application of voltage gradient perpendicular to the direction of the
initial electrophoresis the gel, the sample migrates out of the gel and onto the filter paper; this is
essentially the standard transfer methodology for western blot. After transfer, the membrane is
stained with Coomassie Blue R-250 and destained to locate the protein bands. Sections containing
the proteins bands can then be excised for amino acid analysis and N-terminal protein sequencing.

Efficiency of electroblotting is dependent upon structural parameters of the membrane,

including specific surface area, pore size distribution and pore volumes. Almost quantitative
retention (>90%) of proteins was obtained for membranes with a high specific surface area and
narrow pores.

II. OBJECTIVE

• To transfer protein samples electrophoretically from polyacrylamide gel to PVDF

membrane.

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III. MATERIALS & METHODS

MATERIALS:

• 1× electroblotting transfer buffer

• Polyacrylamide gel containing proteins of interest
• 100% methanol
• Powder-free gloves
• Test tube
• Electro blotting apparatus
• PVDF transfer membrane
• Fiber pad
• Whatman no. 1 filter paper
• Trays
• Magnetic Stirrer
• Power supply

METHODS:

1. Wet PVDF with Methanol for a few seconds.

2. Then transfer the PVDF membrane from the methanol to a dish containing 1x
electroblotting transfer buffer and keeps for 5 minutes.
3. Dip two pieces of filter paper in 1x Electroblotting transfer buffer (above the PVDF
membrane).
4. Soak the fiber pads in blotting buffer.
5. Remove gel for the electrophoresis cell and soak in Electroblotting buffer (above the fiber
pad) for 15 minutes.
6. Then assemble the transblot unit.
7. For that Place the opened gel holder cassette on a clean, flat surface. Place one transfer
buffer–wetted fiber pad on the top surface, followed by the filter paper.
8. Place the gel face down on the support, so that the cut edge is now on the right-handside.
9. Remove wet PVDF membrane from the tray containing transfer buffer and place above the
gel. Roll a test tube across the membrane to avoid air bubbles between membrane and
gel.
10. Place the other wetted filter paper above the PVDF membrane.
11. Place the other fiber pad on top of the membrane and close the holder.
12. Slide the assembled transfer cassette into the tank with the gel on the cathode side and the
membrane on the anode side.
13. Place the Electroblotting apparatus above the magnetic stirrer. Drop the stir bar.
14. Fill the transfer tank with 1× transfer buffer so that the buffer completely covers
theElectrode panels but does not touch the electrical connectors.
15. Turn on the Magnetic stirrer. Assemble the sandwich and electroblot at either constant
voltage of 50Volts at room temperature for 30 minutes or constant current of 500 mA for
the same amount of time.

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16. At the end of the transfer period, turn off the power and disconnect the power supply. Open
the transfer sandwich and remove the membrane and gel. Thoroughly rinse the membrane
with high-purity water, three times, and 5 min each.

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 Week 8: Laboratory Exercise 8
Electroblotting from Polyacrylamide Gels

Name: Date: Points:

I. ABSTRACT
Protein transfer from polyacrylamide gels to retentive membranes is now primarily
used for immunoblotting. The use of Edman chemistry for electroblotting proteins for N-
terminal and internal sequencing was a second application. This unit contains procedures
for electroblotting proteins from polyacrylamide gels onto various membranes, including
PVDF and nitrocellulose. Protocols for electroblotting using semidry and dry systems are
provided in addition to the commonly used tank or wet transfer system. For specialized
applications, this unit also describes procedures for eluting proteins from membranes
using detergents or acidic extraction with organic solvents.

II. INTRODUCTION

Electroblotting proteins from polyacrylamide gels onto retentive membranes is

commonly used to facilitate protein identification and characterization procedures. It is
primarily used for immunoblotting, but it was previously widely used for protein isolation
prior to N-terminal protein sequencing. This latter application is still useful for specialized
applications such as defining the N-terminus of protein fragments or analyzing proteins with
poorly defined genomes. Most previous Edman sequencing applications involved proteomics
analysis using mass spectrometry. In some cases, stained blots are only used to identify
protein band patterns, leaving the gel unaltered for the next steps. For applications requiring
proteins in solution, the protein of interest can be eluted from the membranes as an
alternative to direct elution of the protein of interest from the polyacrylamide gel. The
electroblotting methods in this unit require that the proteins have already been separated on
a polyacrylamide gel

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 III. METHODS
For a few seconds, wet PVDF with Methanol. The PVDF membrane is then transferred
from the methanol to a dish containing 1x electroblotting transfer buffer and left for 5
minutes. 1x Electroblotting transfer buffer dipped two pieces of filter paper (above the PVDF
membrane). Blotting buffer should be applied to the fiber pads. Remove the gel for the
electrophoresis cell and soak it for 15 minutes in Electroblotting buffer (above the fiber pad).
After that, put together the transblotting unit. To do so, place the opened gel holder cassette
on a clean, flat surface. On the top surface, place one transfer buffer–wetted fiber pad,
followed by the filter paper. Place the gel face down on the support, with the cut edge now on
the right.

IV. RESULTS/ANALYSIS

Semi-dry blotting, a variant of this technique, requires only a small amount of buffer to
saturate the membrane and blotting papers, establishing electrical contact between the gel
and the apparatus itself. The benefit is that low voltage and current are required for transfer,
eliminating the need for a high current power supply. Electroblotting can be done with either
nylon or nitrocellulose. Because electroblotting generates heat, cool the electrolyte buffer
before use and keep the temperature under control during the run. The efficiency of
electroblotting is determined by membrane structural parameters such as specific surface
area, pore size distribution, and pore volumes. Protein retention was almost quantitative
(>90%) for membranes with a high specific surface area and narrow channel.

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 V. DISCUSSION AND CONCLUSIONS

To use this technique for amino acid analysis or N-terminal sequencing, the proteins must be
transferred to a membrane that is resistant to the chemicals used in these analytical procedures.
Proteins are transferred to a chemically stable membrane, polyvinylidene difluoride, for sequencing
and amino acid analysis (PVDF). PVDF membranes bind proteins primarily through hydrophobic
interactions and are widely used due to their chemical and physical stability. Electrophoretic transfer
is accomplished by placing the gel adjacent to the membrane in a special cassette, which is then placed
in an electrophoretic buffer tank (tank electroblotting). The PVDF membrane is "activated" by dipping
it in methanol and then placing it in methanol-containing transfer buffer. The gel-PVDF sandwich is
placed in a custom-made holder, which is then placed in the buffer-containing electrophoresis unit.
The sample migrates out of the gel and onto the filter paper when a voltage gradient perpendicular to
the direction of the initial electrophoresis is applied to the gel; this is essentially the standard transfer
methodology for western blot. Transferring proteins from polyacrylamide gels to blot membranes has
numerous advantages. Transferred proteins can be eluted from the membrane, probed with antibodies
(immunoblotting), sequenced at the N-terminus, or stained using a variety of highly sensitive
techniques. Furthermore, by loading samples in multiple lanes on the gel before electroblotting,
multiple manipulations can be performed on the same blot. This unit previously covered in situ
protease or chemical cleavage of proteins for further structural analysis.

VI. LITERATURE CITATION

Dunn, M. J., Dunn, M. J., Dunn, M. J., Gravel, P., Dunn, M. J., Akins, R. E., Tuan, R. S., Serra, P.,

Dunn, M. J., Gravel, P., Dunn, M. J., Akins, R. E., Tuan, R. S., Serra, P., Dong, W.,

Gooderham, K., Hayes, C. A., Sackstein, R., Fuhlbrigge, R., . . . Fuhlbrigge, R. (2009).

Electroblotting of Proteins from Polyacrylamide Gels for Chemical Characterization |

Springer Nature Experiments. MJ Dunn.

https://experiments.springernature.com/articles/10.1385/0-89603-353-

8:37?error=cookies_not_supported&code=8273a7d7-cb60-4f73-aca4-dae6967b1683

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VII. REVIEW QUESTIONS

1. Electroblotting is the transfer of onto a membrane.

a. Proteins
b. Carbohydrates
c. Lipids
d. None of the above

2. Transfer of the proteins can be carried out using?

a. Vaccum
b. Capillary field
c. Electric field
d. All the above

3. It is a necessity that the membrane is located between the gel and the cathode, as the current and
sample will be moving in that direction.

a. True
b. False

4. PVDF is

a. Chemically stable
b. A plastic material
c. Both a and b
d. None of the above

5. The single most important factor in efficient electroblotting is the amount of present in the
gel during electrotransfer.

a. SDS
b. Protein
c. Acrylamide
d. TEMED

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 Week 8: Laboratory Exercise 8
Electroblotting from Polyacrylamide Gels

THESE SECTION TITLES SHOULD BE USED TO LABEL EACH SECTION OF YOUR REPORT

I. Title – Descriptive to-the-point sentence describing your study.

II. Abstract – Summarize the report in no more than 200 words. Someone pressed for time should be
able to read just the abstract and understand the whole experiment from rationale to the interpretation.

III. Introduction – Provide some context for your simulation experiment. What will the experiment tell
you? What is your hypothesis? Why are you doing this experiment in the first place? Provide some
background information to set the stage. Remember you are focusing on the local data. What question
were you trying to answer with this data? How did you analyze this data? You can search for papers that
support your experiment (Google Scholar).

IV. Methods – In this section of the report, you should present the steps that were followed in your
simulation experiment. Do not simply copy the protocol from your stated presentation. Remember to
include information on the process even though you didn’t do this part and remember to imagine that
you did processes explained to other students. Make sure to cover all the phases of the simulation
experiment. You don’t have to detail the amounts of each buffer you used but focus more on the steps
you performed and why. Don’t forget to include the method used to characterize the sites and the
statistical test you performed.

V. Results/ Data Collection/ Analysis – All the data that was theoretically collected during the
experiment should be presented in a data table or tables). Graphs (if necessary) should be shown here
and should be labeled appropriately. Remember if someone off the street looked a graph, they should be
able to understand what it represents. Don’t just report the numbers. This is the place where you should
describe your results in the context of the simulated experiment you performed and say something
about what the numbers mean. Also remember to report what statistical test you used and the results of
that test if there is any.

VI. Discussion/ Conclusions – This portion of the report is used to clearly explain whether the results
support or refute the hypothesis being tested. Explain what your findings mean and what conclusions
you can draw from the data. Don’t just repeat your data, this is where you need to demonstrate that you
understand what your simulated experiment means biologically. Think in terms of biotechnology or
molecular biology and talk about your results with the big picture in mind. Sources of error and
suggestions for improvement should be included in this section. Don’t forget to go back to the big
question and address any implications you see your simulated experiment having. You need to convince
us that your work is relevant to the big picture. You can include citations for papers here. These papers
could be ones that you found with different methods or papers that found similar results.

VII. Literature Citation – You should use at least 2 outside sources, particularly in your introduction
and discussion section. These sources may provide background information or may support your
conclusions. Make sure to use proper citation format and make it consistent. Much of what is related in
discussion and the lab protocols can be found by a simple online search. You should endeavor to find a
primary source rather than try to site the laboratory protocol as your source

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 Week 8: Laboratory Exercise 8
Electroblotting from Polyacrylamide Gels

Name: Date: Points:

Report Item Point Rating and Comments

System
Title 1
• Reflects overall research 1
purpose/question
Abstract 3
• ≤200 words 1
• Background/questions, methods, 2
results, and conclusions each
summarized briefly
Introduction 5
• Appropriate background 1
information 2
• Describe the general purpose of the
experiment 3
• Address how this study contributes
to big picture
Methods 5
• Explain each major step/method and 3
how you did each
• Explain why each major step is 2
important to the entire process
Results 7
• Describe results 3
• Data recorded in tables/figures 2
• Statistical test used and what it 2
means
Discussion 7
• Summarize results/relevance to big 2
picture
• Conclusion stated/implications 3
discussed
• Improvements recommended 2
Literature Citation 3
• Cited within write up 1
• Citation information correct 1
• 2 outside sources 1
Demonstrated mastery of the concepts 14
Grammar 5
Total Points 50

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