Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology . In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells .
The Western blotting procedure relies upon three key elements to accomplish this task:
Once detected, the target protein will be visualized as a band on a blotting membrane, X-ray film, or an imaging system . Since Western blotting is accomplished rapidly, using simple equipment and inexpensive reagents, it is one of the most common laboratory techniques . The results achieved are also easy to interpret, unique, and unambiguous . Therefore, it is routinely used on its own, or along with other immunoassays, in research and clinical settings.
Western blots are in wide use across a broad range of scientific and clinical disciplines . Their ability to clearly show the presence of a specific protein both by size and through the binding of an antibody makes them well-suited for evaluating levels of protein expression in cells, and for monitoring fractions during protein purification . Likewise, they are helpful for comparing expression of a target protein from various tissues, or seeing how a particular protein responds to disease or drug treatment .
In many cases, Western blots are used in combination with other key antibody based detection techniques, such as ELISAs or immunohistochemistry.
In these instances, Western blots provide confirmation of results both in research and diagnostic testing . For example, with HIV and prion disease, Westerns are used as a key supplemental screen since their results are less ambiguous, and quicker, than other methods . Moving forward, Western blots continue to be of value in confirming results from antibody arrays, making them suitable for use in proteomics research.
Antibodies selected for immunodetection should be Western blot tested if possible, with attention paid to the experimental conditions recommended by the antibody supplier . Usually, Western blot positive antibodies recognize a short linear sequence of amino acids found within the target protein that remains intact, or becomes visible, when the target protein is fully unraveled . This is because most Western blots are carried out under denaturing and reducing conditions which remove all higher order protein structure .
In contrast, some epitopes can be conformational, forming a three dimensional structural configuration of amino acids that will be lost upon denaturation of the protein . Thus, not all antibodies work in a typical Western blot . Since Western blot procedures allow for flexibility in choosing gel electrophoresis and blotting conditions, it is possible to modify buffers to retain enough higher order protein structure for detection by some antibodies .
AbD Serotec is one of the world’s leading manufacturers of primary and secondary antibodies for Western blot anaylsis. In addition, we offer ISO-certified pre-made buffers to simplify immunostaining and make results more consistent from blot to blot. Our 100% Satisfaction Guarantee is backed by our quality, which makes ordering from AbD Serotec totally risk free!
Our range of specialized reagents are designed to significantly improve results in Western blotting:
Block Ace This high-performance blocking reagent for Western blotting is superior to 1% BSA. In comparison to BSA, Block Ace provides reduced backgrounds and sharper standard curves. It can also be used as a wash buffer, and for diluting antibodies.
HiSpec This assay diluent works to significantly reduce cross-reactivity and non-specific binding in Western blotting, greatly improving your results. HiSpec assay diluent is designed to be used instead of sample buffer or antibody dilution buffer within the Western blotting protocol.
AbGuard® HRP Stabilizer Is ideal for use as a diluent for peroxidase conjugated antibodies and other peroxidase conjugated proteins. It provides superior stabilization of HRP-conjugates at low and high dilutions, preventing the loss of peroxidise activity.
|Description||Format||Pack Size||Product Code|
|Block Ace||Reagent||20 x 4g||BUF029|
|HiSpec Assay Diluent||Ready-to-use||50ml||BUF049A|
|HiSpec Assay Diluent||Ready-to-use||125ml||BUF049B|
|HiSpec Assay Diluent||Ready-to-use||500ml||BUF049C|
|AbGuard® HRP Stabilizer||Ready-to-use||1000ml||BUF060C|
|AbGuard® HRP Stabilizer||Ready-to-use||100ml||BUF060B|
|AbGuard® HRP Stabilizer||Ready-to-use||500ml||BUF060A|
AbD Serotec offers a range of antibodies to popular loading controls suitable for Western blotting. Our loading control antibodies are offered in a variety of different formats ideal for saving time with direct staining techniques and for use in infrared imaging systems e.g. LI-COR Odyssey®.
Horseradish-peroxidase conjugated antibodies are preferentially used whenever an indirect method is used for detection on Western Blots. AbD Serotec supplies a range of species specific antibodies recognizing immunoglobulin or any of its isotypes. Monoclonal and polyclonal secondary antibodies are available, as a whole IgG or a F(ab’)2 fragment. Performance is further enhanced by affinity purification for all STAR reagents and many anti-mouse, -rat, and -hamster secondary antibodies are multi-species adsorbed.
|Goat Anti Mouse IgG (H&L):Alk. Phos. (Multi Species Absorbed)||Alk Phos||0.5mg||STAR117A|
|Goat Anti Mouse IgG (H&L):HRP (Multi Species Absorbed)||HRP||0.5mg||STAR117P|
|Donkey Anti Sheep/Goat IgG:Alk. Phos.||Alk Phos||1mg||STAR88A|
|Donkey Anti Sheep/Goat IgG:HRP||HRP||1ml||STAR88P|
|Sheep Anti Rabbit IgG:HRP (Multi Abosrbed)||HRP||1mg||STAR54|
|Donkey Anti Rat IgG (H&L):Alk. Phos. (Mouse Absorbed)||Alk Phos||1ml||643004|
|Goat Anti Rat IgG (H&L):HRP||HRP||2mg||5204-2504|
LI-COR Odyssey® is a registered trademark of LI-COR Biosciences, Inc. USA.
AbGuard® is a registered trademark of MorphoSys UK Ltd