Western blotting questions?

Western blotting questions?

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everyone. I've just been introduced to the procedure of Western blotting from my reading, though I'm not entirely sure about some points. I'd appreciate it if someone could help me with this.

What exactly is the function of the blocking buffer?

Why does one need a secondary antibody?

When discussing nitrocellulose membranes, say of a 0.45 5m pore size, what does 5m refer to?

Blocking buffer

Once the proteins in the gel have been transferred to the nitrocellulose membrane it is necessary to coat the rest of the surface of the membrane with an unrelated protein. This is necessary because all proteins will bind non-specifically to the nitrocellulose. Once the membrane has been blocked the only way that antibiody proteins (the probe) will bind to the membrane is via the transferred proteins that they recognise. The usual source of a blocking protein is defatted milk powder, i.e. casein, although some people use (or used to use) bovine serum albumin.

Secondary antibody

In a typical blotting experiment you expose first with a primary antibody directed against the protein of interest, for example this might be an IgG raised in a rabbit. Then in a second step you add a secondary antibody which will bind to the first one - in this case this might be goat ant-rabbit IgG (i.e. anti-rabbit IgG raised in a goat). Why is this done? The secondary antibody is in the form of an antibody-enzyme conjugate, typically with horseradish peoxidase, and the final development of the blot detects the presence of the conjugated enzyme.

Why use a secondary antibody? Two reasons: - firstly, there will sometimes be a degree of amplification if more than one secondary antibody molecule binds each primary antibody molecule; but more important is that it means that it is not necessary to produce an enzyme conjugated form of every primary antibody, which is time-consuming and quite tricky. So in your work you may be studying several different proteins using Westerns, but you will be able to use the same secondary antibody to detect all of them.

Pore size

That should be 0.45 - 0.5 µm. Pore sizes are measured in micrometres.

15: Western blots

  • Contributed by Clare M. O&rsquoConnor
  • Associate Professor Emeritus (Biology) at Boston College

Western blots are one of the most widely used techniques in cell biology. In a western blot, investigators take advantage of the exquisite sensitivity of antibodies to identify proteins of interest in complex samples. In this lab, you will learn about the different kinds of antibodies used in western blots. You will use western blots to analyze Met and LacZ protein expression in your transformed yeast strains.

At the end of this lab, students will be able to:

  • Explain how monoclonal and polyclonal antibodies are produced.
  • Identify the different functional regions of antibodies and explain how they are used in western blots
  • Design a strategy that uses antibodies to detect epitope-tagged proteins.
  • Prepare a western blot to analyze protein expression in cell extracts.

Western blots provide a method to find the proverbial &ldquoneedle in a haystack.&rdquo A typical cell expresses thousands of different proteins, and it is often difficult to detect changes in expression of your favorite protein (Yfp) without a probe that is capable of discriminating the Yfp against a large background of unrelated cellular proteins. Fortunately, antibodies provide highly specific molecular probes that can be used to detect the expression of proteins on western blots. To appreciate the sensitivity of western blots, it&rsquos helpful to have some understanding of antibody structure and antibody production during immune respones. (Disclaimer: The following paragraphs provide a highly abbreviated overview of antibodies and one segment of the complex vertebrate immune system. The Department offers an immunology course that will introduce you to the finer details of this fascinating system.)

Western blot: technique, theory, and trouble shooting

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

Keywords: Bio-medical research protein western blot.

Conflict of interest statement

Conflict of Interest: None declared.


Assembled rack for gel solidification

Assembled rack for gel solidification

Add gel solution using a transfer pipette

Add running buffer to the…

Add running buffer to the electrophorator

Add samples and molecular marker…

Add samples and molecular marker to the gel, after removing the combs

(a) Samples running through the…

(a) Samples running through the stacking gel (lower voltage). (b): Samples running through…

Primary antibodies, secondary antibodies and controls for western blotting

The primary antibody usually recognizes a specific protein or epitope and is conjugated or tagged to a fluorescent dye or an enzyme to enable subsequent detection. The tagged antibodies are detected directly (direct western blotting) or using secondary antibodies (indirect western blotting). Depending on the method, secondary detection is carried out using antibodies conjugated to enzymes or fluorophores. Horseradish peroxidase (HRP) is the most common enzyme used for tagging.

These fluorophore-conjugated antibodies utilize the property of fluorophores to absorb light at a certain wavelength and emit it at a different wavelength. By using different combinations of filters, specific wavelengths of light can be measured. Since fluorophores with absorption and emission maxima spanning the entire light spectrum are available, combinations of antibodies conjugated to fluorophores of different wavelengths can be used to detect multiple proteins.

Labeled antibodies against several important biological target proteins are commercially available.

Traditional Immunodetection


  • Primary and Secondary Antibodies
  • Orbital shaker* (Product No. Z768499)
  • Troughs 2-3 mm depth, slightly larger than the size of your blot
  • Detection substrates (for use with peroxidase- or phosphatase-antibody conjugates)
    - Chemiluminescence substrates
    - Chromogenic substrates

*Shaker and Troughs only required for traditional immunodetection for rapid, vacuum-driven immunodetection using the
SNAP i.d. ® system, refer to the SNAP i.d. ® immunodetection protocol.

  1. Place the blot in the blocking solution and incubate with agitation for 1 hour.
  2. Place the blot in the primary antibody solution, for example Monoclonal Anti-Beta-Actin Antibody and incubate with agitation for 1 hour. The solution should move freely across the surface of the membrane.
  3. Place the blot in PBS and wash for 10 minutes. Repeat twice with fresh buffer.
  4. Place the blot in the secondary antibody solution and incubate with agitation for 1 hour at RT or 37 °C.
  5. Place the blot in PBS and wash for 10 minutes. Repeat twice with fresh buffer.
  6. Proceed with chromogenic, chemiluminescent, or fluorescent detection. Also view related resources for protein detection:
    Chromogenic and Chemiluminescent Detection of Proteins
    Immunodetection Using BCIP/NBT Substrate

Do you know who invented the Western blot?

1979 certainly was a busy year in the development of western blotting (also called "Protein blotting" or "Protein immunoblot"). They were all working hard and trying to publish.

But who deserves the real credit?

Most researchers know western blotting evolved from Southern blotting (Ref 1), invented by Edwin Southern at University of Edinburgh in 1975, then northern blotting (Ref 2), invented by George Stark's Stanford group in 1977.

All 3 groups of researchers in Seattle, Stanford, and Basel seem to have been working independently from 1977 to 1979 on a better method to detect proteins using antibodies. They all attempted to publish in 1979 and certainly all deserve some credit for the development of the western blot. But who should we cite as the real inventor? This question is not as easy to answer, but it is a very interesting story.

George Stark's group at Stanford University, including Jaime Renart & Jakob Reiser, published first (Ref 3), submitting in April 1979 and published in July 1979. They used passive transfer of the proteins, then 125-I-labeled Protein A for detection.

Harry Towbin's group in Basel, Switzerland, including Towbin & Julian Gordon (at Friedrich Miescher Institut) and Theophil Staehelin (at Hoffmann-La Roche), submitted in June 1979 and was published in September 1979 (Ref 4). They developed the method of electrophoretic transfer of proteins to membranes (instead of passive transfer like Stark) and the now-ubiquitous immunoblotting sandwich using electric current to transfer proteins from the SDS-PAGE gel to the membrane (Ref 5). Their procedure also used secondary antibodies for detection thus, this appears to the actual method that is generally followed for westerns.

W. Neal Burnette, working in Robert Nowinski's lab at the Fred Hutchinson Cancer Research Center in Seattle, submitted in 1979, but it was rejected, then eventually published in 1981 (Ref 6). Burnette definitely gave the technique the name "Western blotting" as a nod to Southern blotting and because their lab was on the west coast. He developed his technique independently, including the electrophoretic transfer step, but became aware of Stark's and Towbin's publications before he submitted his in 1979. He still believed his version was simpler and more universal, thus worthy of publication (Ref 7). He mentions using "unmodified nitrocellulose, radiolabeled Protein A detection, and 2-D separations" as advantages (Ref 7). He also said his method "simplified antigen transfer, antibody detection, and complex visualization" (Ref 8). Interestingly, Burnette reports that his submission in 1979 was "dismissed - not on the basis of the Towbin publication but because the technique was transparently obvious, made no fundamental contribution to the scientific literature, and, most damning, had been given the flippant name of 'Western blotting' in honor, of course, of Edwin Southern and of the West Coast location of its invention." (Ref 8). His procedure was finally published in 1981 after it had already become famous through distribution of pre-prints to friends, who repeated the process (Ref 9). "Pretty soon, I was running a daily seminar on blotting by telephone. I was talking to everyone. because they couldn't read the Xeroxed copy they had. [Western blotting] was taking up all my time by talking on the phone." (Ref 9).

So, who actually invented the western blot and should be given credit for it?

Stark's group published first. Towbin's group developed what appears to be the most common method, including the electrophoretic transfer method and buffers, as well as the use of secondary antibodies. Burnette gave the technique the name among other modifications and was very instrumental in popularizing western blotting. In a 2005 Nature Methods Classic Protocol (Ref 10), Michael Eisenstein wrote "It is arguable. that Burnette's most significant contribution to immunoblotting came when he christened the technique 'western blotting'". When many researchers cite western blotting, they often will reference both Burnette's and Towbin's papers, so perhaps the most accurate answer is that both of them should get credit, but we leave it up to you to answer the question for yourself.

We hope you enjoyed this story and the background behind a technique many of you use every day in your lab. We always find it fascinating to learn more about the history of techniques that have become so vital for exciting and life-saving discoveries around the world. For additional background information and more interesting stories and insight into the development of this procedure, we recommend the references below.

The information presented here is accurate to the best of our knowledge, but we welcome any feedback you might have as to your thoughts on the subject, your experiences/personal knowledge, or any other insight - there is always more to the story than what gets published in the public domain. You can contact us by email at [email protected].

GenHunter has several popular product lines for Western blotting, all with the goal of saving you money and making Western blots simple:

  • Our PerfectWestern Containers are available in 38 different sizes and have ultra-smooth flat surfaces, lids, and 90 degree edges to perfectly accommodate Western blots of all sizes, significantly reducing the amount of antibodies needed (often by 75%), while still giving beautiful blots. You can download our PerfectWestern catalog here.
  • The PerfectMembrane is available in 3 different standard gel sizes, in 4 materials (3 types of PVDF and Nitrocellulose), and is generally about half the cost of other pre-cut membranes.
  • Our PerfectFilm is a high-quality, especially sensitive blue autoradiography film that is ideal for most applications including Western blots using ECL substrates for HRP or AP!
  • The PerfectRocker offers the standard back-and-forth rocking motion preferred by many labs for Western blots. It also includes adjustable speed (2 to 30 RPM) and adjustable pitch (0 to 30 degree tilt angle).
  • The new PerfectWaver, Belly Dancer, and Belly Button Orbital Shakers have a unique 3D undulating orbital motion that is ideal for Western blots, but also work for many other applications. These shakers are extremely durable and allow perfect membrane movement.

1) Southern, EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. (1975) Journal of Molecular Biology 98:503-517.
Click here to see the original Southern blotting reference

2) Alwine JC, Kemp DJ, Stark GR: Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. (1977) PNAS 74:5350-5354.
Click here to see the original northern blotting reference

3) Renart J, Reiser J, Stark GR: Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. (1979) PNAS 76:3116-3120.
Click here to see George Stark's western publication

4) Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. (1979) PNAS 76:4350-4354.
Click here to see Harry Towbin's western publication

5) Towbin H: Origins of Protein Blotting. (2009) Protein Blotting and Detection: Methods and Protocols, Biji T Kurien and R Hal Scofield (eds.) Methods in Molecular Biology. 536:1-3. Humana Press/Springer, New York, NY.
Click here to see Towbin's 2009 book chapter

6) Burnette WN: "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. (1981) Analytical Biochemistry 112:195-203.
Click here to see Neal Burnette's western publication

7) Burnette WN: Western blotting: Remembrance of Things Past. (2009) Protein Blotting and Detection: Methods and Protocols, Biji T Kurien and R Hal Scofield (eds.) Methods in Molecular Biology. 536:5-8. Humana Press/Springer, New York, NY.
Click here to see Burnette's 2009 book chapter

8) Burnette WN: Western blotting. Clinical Chemistry (2011) 57:132-133.
Click here to see the 2011 'Citation Classic' commentary

9) Mukhopadhyay, R: W. Neal Burnette: The man behind the Western blot. ASBMB Today. (January 2012) pg 17-19. The American Society for Biochemistry and Molecular Biology, Rockville, MD.
Click here to see the 2012 ASBMB story on Dr. Burnette

10) Eisenstein, M: A look back: westward expansion. Nature Methods. (2005) 2:796.
Click here to see the 2005 Eisenstein 'Classic Protocol' story

Please contact us if you have any questions or comments.

Applications & Technologies

Western Blotting Techniques

Learn more about western blotting techniques. Find step-by-step protocols and helpful tips on equipment, membranes, transfer conditions, and detection methods.

Protein Separation and Analysis

Bio-Rad's V3 Western Workflow facilitates speed and validation at each step of a western blotting experiment &mdash from running gels to quantifying proteins.

Protein Electrophoresis

Find protocols, video tutorials, and selection guides to help you at every step of your electrophoresis experiments.

Imaging and Analysis

Find information on protein visualization and quantitation methods, gel and blot imaging instrumentation, and image analysis software.

Western Blotting

Western blotting is a well-established analytical technique for detecting, analyzing, and quantifying proteins. This method is widely used to detect specific protein molecules in complex samples such as tissue homogenates and cell lysates. Western blotting typically involves protein separation by gel electrophoresis followed by transfer to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane. After proteins have been transferred, they can be stained for visualization and directly identified by N-terminal sequencing, mass spectrometry or immunodetection.

In Western blotting immunodetection, proteins are identified through their binding to specific antibodies. Typically, a primary antibody is used in combination with an HRP- or AP-conjugated secondary antibody for chemiluminescent or colorimetric detection using an appropriate substrate. Alternatively, a fluorescently labeled primary or secondary antibody can be used for direct visualization.

Western blotting is used extensively in biochemistry to detect the presence of specific proteins, to determine the extent of post-translational modifications, to verify protein expression in cloning applications, to analyze protein and biomarker expression levels, in antibody epitope mapping, and to test for markers of disease in clinical settings.

The need to simultaneously analyze more proteins in limited samples has driven ongoing research into improving the sensitivity and speed of blotting techniques. Double blotting eliminates false positives caused by nonspecific interactions. Far-Western blotting enables the detection of specific protein-protein interactions. Southwestern blotting is used to identify proteins that interact with specific DNA sequences. Multistrip blotting increases throughput while minimizing inter-blot variability. New technologies are being developed to reduce the amounts of protein required to produce a signal and improve the quantitative capabilities of Western blotting.

The Journal of Biological Chemistry

Requires publishing statistical analysis of experimental findings

“Statistical analyses of variation and precision for establishing differences between experimental groups should be preferably reported using the standard deviation (SD) or confidence intervals (CI). 2,3 ”

Statistical analyses are useful to interpret and validate results

Using Mean ± SEM bar graphs can be misleading and may alter the interpretation of data.

LI-COR can help you generate statistically significant data for quantitative Western blot analysis

Get detailed information on data interpretation, and calculation of coefficient of variation (% CV), arithmetic mean (average), and standard deviation for normalization and quantitative values in the analyses sections of our protocols.

With these products, protocols, and tools – and LI-COR’s +17 years of Western blotting experience – we can help you be successful regardless of where you are planning to publish your data.

Note: These resources should also assist you with other journals’ requirements but be sure to check guidelines for the journal to which you are submitting your publication.

Learn more about how publication requirements may affect you.


    Nature. Macmillan Publishers Limited, 2016. Web. 31 July 2017. The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 31 July 2017. The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 9 May 2018. Cell. CellPress. Wevb. 31 July 2017. The Journal of Cell Biology. The Rockefeller University Press. Web. 31 July 2017. Science. American Association for the Advancement of Science. Web. 31 July 2017.

Compare protein expression accurately and reduce variability wherever possible.

Protein comparison means taking the definition of quantitative a bit further. It’s possible to get two quantitative Western blots and compare them inaccurately&hellip

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Western blot analysis

TBS buffer 20 mM Tris-HCl, pH 7.5
150 mM NaCl TBS-T buffer 20 mM Tris-HCl, pH 7.5
150 mM NaCl
0.05% Tween 20 Sample buffer 1%(w/v) Triton X
4 M Urea
50 mM Tris-HCl, pH 6.8
150 mM NaCl
2% (w/v) SDS
10% (w/v) glycerol
2% (w/v) mercaptoethanol Blocking Solution 5% nonfat dry miik (included in detection kit, Bio-Rad, Cat.#170-6404) in TBS-T
TBS-T buffer

Precast Gels (7.5% Ready Gel Precast Gels, Bio-Rad, Cat.# 161-1118)

Mini Trans-Blot cell (Bio-Rad, Cat.# 170-3930)

Immun-Blot PVDF (Bio-Rad, Cat.# 162-0174)

Immun-Star TM Goat Anti-Mouse HRP Detection Kit (Bio-Rad, Cat.#170-5044)

Primary Antibody Anti-Renilla Luciferase (clone 5B11.2, Chemicon International, )
The dilution for primary antibody is1:2,000 from a stock solution Secondary Antibody Immun-Star TM Goat Anti-Mouse-HRP Conjugate (Bio-Rad, Cat.#170-5047)
The dilution for the secondary antibody is 1:30,000 dilution

    Collect protoplast suspensions from 4 wells in a 96-well plate (a total of