What is Western Blotting?

Western Blotting

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:

  • the separation of protein mixtures by size using gel electrophoresis;
  • the efficient transfer of separated proteins to a solid support;
  • and the specific detection of a target protein by appropriately matched antibodies.

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.


Why use a Western Blot?

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.


Western Blotting Antibodies

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!


Western Blotting Buffers & Accessories

  • Product Code: BUF029|BUF049A|BUF049B|BUF049C|OBT7271|OBT7272|BUF060C|BUF060B|BUF060A|BUF036A
DescriptionPack SizeApplicationsPriceQuantity
10X Phosphate Buffered Saline1 LitreIY
BUF036A
AbGUARD® HRP Stabilizer100 mlC, E, P, WB
BUF060A
AbGUARD® HRP Stabilizer1000 mlC, E, P, WB
BUF060C
Block ACE20 X 4gE, WB
BUF029
Hispec Assay Diluent125 mlIY
BUF049B
Hispec Assay Diluent50 mlIY
BUF049A
Hispec Assay Diluent500 mlIY
BUF049C
Horseradish Peroxidase25 mgE, WB
OBT7272

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.


Western Blotting Loading Control Antibodies

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®.

Loading Control Specificity


Secondaries

  • Product Code: STAR117A|STAR117P|STAR88A|STAR88P|STAR54|643004|5204-2504
DescriptionPack SizeApplicationsPriceQuantity
Donkey anti Rat IgG (H/L):Alk. Phos. (Mouse Adsorbed)1 mlC, E, WB
643004
Donkey anti Sheep/Goat IgG:Alk. Phos.1 mgC, E, WB
STAR88A
Donkey anti Sheep/Goat IgG:HRP1 mlC, E, P, WB
STAR88P
Goat anti Mouse IgG (H/L):Alk. Phos. (Multi Species Adsorbed)0.5 mgE, WB
STAR117A
Goat anti Mouse IgG (H/L):HRP (Multi Species Adsorbed)0.5 mgE, WB
STAR117P
Goat anti Rat IgG (H/L):HRP2 mgC, E, WB
5204-2504
Sheep anti Rabbit IgG:HRP (Multi Adsorbed)1 mgC, E, WB
STAR54

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.


LI-COR Odyssey® is a registered trademark of LI-COR Biosciences, Inc. USA.
AbGuard® is a registered trademark of MorphoSys UK Ltd