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

  • Ordinal Number: 9015
DescriptionPack SizeApplicationsPriceQuantity
AbGUARD® AP Stabilizer100 mlC, E, P, WB
BUF053A
AbGUARD® HRP Stabilizer Plus100 mlC, E, P, WB
BUF052A
AbGUARD® HRP Stabilizer Plus1000 mlC, E, P, WB
BUF052C
AbGUARD® HRP Stabilizer Plus500 mlC, E, P, WB
BUF052B
Block ACE20 X 4gE, WB
BUF029
Horseradish Peroxidase25 mgE, WB
OBT7272
Protein Stabilizer100 mlIY
BUF069A
Protein Stabilizer1000 mlIY
BUF069B

 

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®.

Western Blotting Loading Control Antibodies

  • Ordinal Number: 9021
DescriptionPack SizeApplicationsPriceQuantity
Human anti Cyclophilin A:Biotin0.1 mgE, WB
HCA005B
Human anti Cyclophilin A:DyLight®6800.1 mgWB
HCA005D680
Human anti Cyclophilin A:DyLight®7500.1 mgWB
HCA005D750
Human anti Cyclophilin A:DyLight®8000.1 mgWB
HCA005D800
Human anti Cyclophilin A:HRP0.1 mgE, WB
HCA005P
Human anti Cyclophilin A0.1 mgE, WB
HCA005
Human anti Human Actin beta:DyLight®6800.1 mgF, WB
HCA147D680
Human anti Human Actin beta:DyLight®8000.1 mgWB
HCA147D800
Human anti Human Actin beta:HRP0.1 mgWB
HCA147P
Human anti Human Actin beta0.5 mgWB
HCA147G
Mouse anti Human Actin beta0.1 mgC, IF, WB
MCA5775GA
Mouse anti Human Cyclophilin A:Preservative Free0.1 mgWB
MCA3350Z
Mouse anti PCNA0.1 mgC, F *, IP, P, WB
MCA1558
Mouse anti Porcine Tubulin beta (N-Terminal)0.1 mgC, E, WB
MCA2703
Mouse anti Rabbit GAPDH:Biotin0.1 mgC, E, IF, IP, WB
MCA4739B
Mouse anti Rabbit GAPDH:DyLight®6800.1 mgWB
MCA4739D680
Mouse anti Rabbit GAPDH:DyLight®7500.1 mgWB
MCA4739D750
Mouse anti Rabbit GAPDH1 mgC, E, IF, IP, WB
MCA4739G
Rabbit anti GAPDH (C-Terminal)50 µgWB
AHP996T
Rabbit anti Human VDAC150 µgE, P *, WB
AHP1415
Rabbit anti PCNA50 µgE, P *, WB
AHP1419
Rat anti Tubulin alpha:DyLight®7500.1 mgWB
MCA77D750
Rat anti Tubulin alpha:DyLight®8000.1 mgWB
MCA77D800
Rat anti Tubulin alpha:HRP0.1 mgC, E, WB
MCA77P
Rat anti Tubulin alpha0.5 mgC, E, IF, IP, R, WB
MCA77G

Western Blotting Secondary Antibodies

  • Ordinal Number: 119001
DescriptionPack SizeApplicationsPriceQuantity
Donkey anti Rabbit IgG (H/L) (Human/Rat/Mouse Adsorbed)1 mgC, E
644001
Goat anti Chicken IgA:FITC1 mgC, F
AAI28F
Goat anti Chicken IgG (Fc):HRP1 mgE
AAI29P
Goat anti Human IgA alpha Chain:Alk. Phos.1 mgC, E, WB
5104-2104
Goat anti Human IgA:HRP1 mlE, P, WB
STAR141P
Goat anti Human IgG F(ab')2:HRP0.5 mlE, WB
0500-0099
Goat anti Human IgG/A/M:HRP2 mgE, WB
STAR90P
Goat anti Human IgM:Biotin1 mgE, F, IB
202008
Goat anti Human Lambda Light Chain:RPE0.5 mgF
207009
Goat anti Rabbit IgG/IgM:RPE0.5 mgF
401009
Goat anti Rabbit IgG1 mgE, IB
403001
Goat anti Rat IgM:HRP0.5 mgE, WB
STAR116P
Goat Fab anti Mouse IgG (H/L)1 mgE
0300-0136
Human anti Mouse IgG3:RPE100 TestsF
HCA039PE
Mouse anti Bovine IgG2:HRP0.25 mgE
MCA2441P
Mouse anti Human IgE0.2 mgE
0100-0414GA
Mouse anti Rabbit IgM (B Cell Marker)0.1 mgC, F
MCA812GA
Mouse anti Rat IgA Heavy Chain:FITC0.5 mgF
MCA191F
Mouse anti Rat IgM Heavy Chain:Biotin0.5 mgE
MCA189B
Mouse anti Sheep IgM:FITC0.1 mgF
MCA2223F
Purified Horse IgG10 mgE, ID
PEP001
Rabbit anti Goat IgG (Fab')0.5 mgE
STAR192
Rabbit anti Sheep IgA:Alk. Phos.0.5 mgE
AHP949AB
Sheep anti Bovine IgA:HRP1 mgE
AAI20P
Sheep anti Mouse IgG (H/L):FITC1 mgC
AAC10F

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