Western Blot
Biorbyt dares to do things differently
Western Blot Tips and Tricks
What is Western Blot?
If you need to identify specific amino-acid sequences from a sample, Western Blotting is the technique you should choose. Target proteins are marked using carefully selected primary and secondary antibodies that enable you to visualize the specific protein you are interested in. Western Blotting, like any other laboratory technique, needs to be carried out with precision and understanding at each stage to achieve the best results. With years of experience behind us, we have learned what works best. Biorbyt aim to provide guidance to save you time and frustration and make the process easier and more accurate optimizing your results.
With Western blot, you are aiming for the clearest results via an increased signal-to-noise ratio; displaying clearly the specific bands and minimizing the presence of non-specific bands. There are many steps that can be taken to achieve that goal.
Western Blot Protocol
Western Blotting Protocol in a nutshell:
- Extraction of protein using cell lysis
- Seperation of molecules according to size/weight using electrophoresis
- Transfer molecules to solid membrane
- Incubation with primary and secondary antibodies to visualise target protein by marking
- Washing off unbound antibodies
- Detection of protein of interest
Troubleshooting WB
Non-Specific Binding and Blocking Peptides
Non-Specific Binding
On Western blots, you might sometimes see non-specific binding of an antibody to proteins other than the antigen. This shows itself as multiple bands rather than the 1 or 2 bands you expect. In general, this is more common with polyclonal antibodies, but you sometimes see it with monoclonals. When this happens, it's really important to work out which band or staining is specific to your protein. You can do this by carrying out an immunising peptide blocking experiment.
Blocking Peptides
Before you can run the staining protocol, the antibody has to be neutralised by incubating it with an excess of peptide that corresponds to the epitope recognised by the antibody. These peptides are called blocking peptides. Blocking peptides are the peptides used to raise the original antibody, particularly in the case of polyclonals. You can buy the antibody and the blocking peptide together, as a pair.
The antibody that is bound to the blocking peptide is no longer available to bind to its epitope in the protein on the Western blot or in the cell. The neutralised antibody is then used side-by-side with the original antibody alone, using the original protocol, and the results are compared.
By comparing the staining from the blocked antibody to that of the original antibody alone, you can see which staining is specific. Staining patterns or bands from the blocked antibody will be absent from the Western blot or immunostaining performed with the neutralised antibody
Get your Blocking with Immunizing Peptide (BL) Protocol
Matching Antibodies and Blocking Peptide Pairs
Select our most popular matching peptide pairs below.
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Biorbyt Antibody Conjugates
Biorbyt is now able to offer primary antibodies conjugated to a range of fluorescent and enzymatic labels.
Most protein and cell analysis techniques require the use of primary antibodies and traditionally a labelled secondary antibody for visualisation. However, there are times when directly conjugated primary antibodies have advantages over two step primary/secondary techniques.
For simultaneous detection of multiple target proteins in one experiment, directly conjugated primaries can reduce the nightmare of secondary cross reactivity.
Our conjugated primaries are fully covered by our product guarantee, allowing you peace of mind to experiment with new protocol optimisations. If you are looking for a particular fluorescent conjugate wavelength, please do contact us as we’re keen to allow our offering to make your science work.
Our Primary Antibodies are Conjugated to:
| HRP | CF®488A |
| Biotin | CF®594 |
| FITC | CF®647 |
Antibody Validation
Antibodies are generally raised against either proteins (partial or full length), or a short peptide sequence, specific to their target protein. However, how do you know whether they bind to the target against which they were raised?
Antibody validation is key!
Below are the most basic validation steps that should have been carried out for your antibody:
Peptide raised antibodies - Immunogen validation
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Blast the peptide sequence against the UniProt database to see whether it has identity to its stated target only.
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Any proteins where the identity between peptide sequence and protein sequence for the immunogen is 85% or greater could be detected by that antibody.
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Unfortunately, the exact peptide sequence is not always available, but the amino acid range surrounding the peptide sequence should be. Blasting the region is also helpful.
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All Biorbyt's immunogens are Blasted back to the UniProt database to make sure they detect only the protein(s) they are designed against.
Application validation of antibodies
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If an antibody is said to work for Western Blot, or IHC-P, it should have been tested in that application.
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Raw validation images should be available for the species application stated.
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It is important to see negative as well as positive results.
Additional validations by Biorbyt
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Biorbyt ensure each of its antibodies are tested in Western Blot and IHC paraffin embedded sections in Mouse, Rat and if possible Human tissue sections (although this is sometimes difficult depending on the tissue required for the target).
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Our Western blot images are shown in their raw form, so you can see species and tissues where the antibody did not detect the target, as well as those where it did.
Remember: validation is key! Follow the steps above to find out whether your antibody will bind to the target against which they are raised.
Check out the following Biorbyt protocols:
Antibodies - Chicken Ig Why?
Immunoglobulin Y (IgY) is the dominant immunoglobulin found in avian, amphibian and reptilian species. In 1893, Klemperer1 first described the concept of passive immunity by reporting the presence and transference of tetanus toxin immunity (antibodies) from hen to chick via egg. He noted that antibodies were present in both the blood of the chicken and the yolk of the egg that it laid. However, it wasn’t until 1969 that Leslie and Clem2 coined the term IgY to describe poultry antibodies, including those found in the yolk of the eggs. Until 1969, IgY was known and reported as IgG, but the structural and antigenic differences were shown to be great enough to earn the protein the new Immunoglobulin classification IgY (Figure 1).
(Figure 1) Avian IgY structure, compared to mammalian IgG. Both contain two heavy and two light chains, which consist of a variable domain (VH and VL) and four constant domains (CH1, CH2, CH3 and CH4). IgG has a longer hinge region making it more flexible than avian IgY
IgG and IgY are similar on several levels:
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Both have the characteristic antibody Y shape
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Both contain 2 heavy and 2 light chains
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Both are divalent
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Both play a similar biological role, being the major immunoglobulins to provide defense against infectious agents, appearing in high concentrations after initial synthesis of the higher molecular weight antibody (IgM)
IgY also shares some similarities with mammalian IgE, including similar intrachain disulphide bonding in their extra heavy chain domain. This led to suggestions that IgY may well be an ancestral molecule to both mammalian IgG and IgE, which have since been confirmed by genetic and structural studies3 4 5.
IgG and IgY are different on several levels:
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Little or no immunological cross-reactivity between IgY and mammalian IgG
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IgY - higher molecular weight due to an extra heavy chain constant domain
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IgY lacks a well-defined hinge region
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IgY heavy chains lack Fc domains so they don’t fix complement or bind protein A or protein G
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IgY can facilitate anaphylactic reactions, which can only be done by IgE in mammals
So, given these differences and similarities, why would you want to use IgYs in your research?
The very fact that the molecules are so similar in both their biological role and their structure, yet different enough not to cross-react offers several advantages to a research scientist. In most experimental applications, including Western blotting, immunohistochemistry, immunocytochemistry, ELISA and functional blocking experiments, both IgG and IgY are equivalent, meaning IgY can generally be used as a direct alternative to IgG5.
Advantages of IgY
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Higher avidity - Chickens are not mammals so their reaction to a mammalian antigen is enhanced leading to high-avidity antibodies. The response is particularly to strong to mammalian antigens and especially those which are highly conserved.
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Humane – What could be more humane? The chicken lays the egg, so there is no need to bleed or otherwise conduct procedures on the chicken; you just collect the eggs. Plus, Chickens are cheaper to keep than rabbits!6
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High yield – one chicken can produce large quantities of IgY against low quantities of antigen. Some can produce 2.5 - 3 g of IgY per month, which is 10-20 times the amount of a rabbit6
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Quick – Compared to rabbits, you can obtain high-titre antibody from eggs as early as day 25.
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Lower background - IgY does not bind to IgG Fc receptors meaning you get less false positive staining. They also do not activate mammalian complement systems.
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Cross-reactivity – As there is none to mammalian species, they make an excellent addition to multiple labelling or multi-plex experiments.
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Convenient – IgY is packed neatly in eggs, which can be stored for long periods and the IgY purified to the desired titre/avidity months later.
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IgY can still be digested by papain to produce a divalent Fab fragment.
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IgY can be conjugated to enzymes or fluorophores, biotinylated and gold–labeled by standard procedures, offering you the full range of functionality you would expect from an antibody tool.
So, why not add these fantastic tools to your kit when you next need to visualize a protein? Biorbyt has over 400 Chicken raised antibodies to offer, so check out our website to see if we have added your target to our portfolio.
References
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Klemperer, F. Ueber natürliche Immunität und ihre Verwerthung für die Immunisirungstherapie. Arch. Für Exp. Pathol. Pharmakol. 31, 356–382 (1893).
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Leslie, G. A. & Clem, L. W. Phylogen of immunoglobulin structure and function. 3. Immunoglobulins of the chicken. J. Exp. Med. 130, 1337–1352 (1969).
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Zhang, X., Calvert, R. A., Sutton, B. J. & Doré, K. A. IgY: a key isotype in antibody evolution: IgY antibody. Biol. Rev. 92, 2144–2156 (2017).
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Taylor, A. I., Fabiane, S. M., Sutton, B. J. & Calvert, R. A. The Crystal Structure of an Avian IgY-Fc Fragment Reveals Conservation with both Mammalian IgG and IgE † ‡. Biochemistry 48, 558–562 (2009).
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Lee, W., Syed Atif, A., Tan, S. C. & Leow, C. H. Insights into the chicken IgY with emphasis on the generation and applications of chicken recombinant monoclonal antibodies. J. Immunol. Methods 447, 71–85 (2017).
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Amro, W. A., Al-Qaisi, W. & Al-Razem, F. Production and purification of IgY antibodies from chicken egg yolk. J. Genet. Eng. Biotechnol. 16, 99–103 (2018).
Monoclonal or Polyclonal Antibodies,
The Best Choice for Your Research
Introduction to Antibodies
Plasma cells are specialised B lymphocytes that secrete antibodies (glycoproteins) in response to infection by antigens (toxins or foreign substances). Antibodies, also known as immunoglobulins (Ig), usually have a high affinity to a specific antigen, binding only to one epitope/binding site on an antigen. Monoclonal (Mab) or polyclonal (pAb) antibodies are used in scientific research for a wide range of applications. This article will guide your choice for your research.
Choosing between Monoclonal and Polyclonal Antibodies
Both monoclonal and polyclonal antibodies have specific features, it is important to choose the right antibodies for your research.
- How will the antibodies be used in your research?
- What do you need to achieve?
What are Monoclonal antibodies (mAb)?
Monoclonals are an immortalised hybridoma cell line, a vital research tool since 1975. Monoclonals can detect a single protein or can be used as a probe to locate the protein in vivo. mAbs are created by fusing a single splenic B cell with a myeloma cell, the clones produced are purified from the cells or the lysates and are capable of binding to a single type of epitope. Generation of large enough quantities to purify requires skill and takes up to 12 months, therefore they are more expensive.
What are Polyclonal antibodies (pAb)?
Polyclonal antibodies are derived from different B cells. They can bind to more than one epitope on an antigen. pAbs are created in the serum of an immunised animal, usually goat, sheep, rabbit. The host animal generates multiple B cell clones against every epitope on the antigen. Polyclonal antibodies will have variability across batches, they are quicker and cheaper to produce. This is useful if you are not looking for a specific epitope or your epitopes may have become distorted. Polyclonal antibodies could potentially amplify your signal.
Advantages of Monoclonal and Polyclonal Antibodies in Research
Your choice of monoclonal or polyclonal antibodies will be dictated by the aims of your research.
| Monoclonal Antibodies | |
|---|---|
| Feature | Advantage |
| Specificity for one Epitope | Locating a specific antigen |
| Consistency and binding efficiency | Reproducible, predictable results. Good for assay, therapy production and all diagnostic applications |
| Purified from immortal cell lines | Long term source, stability, repeatability |
| Can be cleaved to reduce nonspecific Fc binding | Useful in flow-cytometry |
| Large quantities of identical antibodies can be produced | Reproducible, predictable results for diagnostic and manufacturing applications |
| Polyclonal Antibodies | |
|---|---|
| Feature | Advantage |
| Recognise and bind to multiple epitopes | More robust for assaying epitopes with slight variations. Useful in Western Blot, IP and ChIP |
| Large quantities easily generated | Detection of target antigen is maximised but can create background signal |
| High antigen detection rate | Useful if consistency over long time periods is not important |
| Can amplify signal | Useful for low target epitope levels |
| Can capture target protein quickly | Useful as capture antibody in Sandwich ELISA and in IP or ChiP |
| Can detect native proteins | Best choice for multiple assay types |
| Can be conjugated with antibody labels easily | Binding affinity is less affected than it would be in mAbs |
Summary
If you require a large quantity of identical antibodies that will bind to one specific epitope, monoclonal antibodies are the best choice. Monoclonals are well suited to diagnostic or manufacturing applications and for therapy and assay production because of their consistency and stability. If it is more essential to increase the chances of detecting your target antigen, choose polyclonal antibodies.
