Western Blotting Principle

What is western blotting, the principle behind western blotting, how western blot works step-by-step, and a detailed WB protocol

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what Is western blot?

Western Blotting separates proteins by size and labels the protein of interest with an antibody

Western blotting (also called Protein Immunoblotting because an antibody is used to specifically detect its antigen) is a widely accepted analytical technique used to detect specific proteins in the given sample. It uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in the given sample (e.g. to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide). The separated proteins are then transferred or blotted onto a matrix (generally nitrocellulose or PVDF membrane), where they are stained with antibodies (used as a probe) specific to the target protein. By analyzing location and intensity of the specific reaction, expression details of the target proteins in the given cells or tissue homogenate could be obtained. Western blotting analysis could detect target protein which is as low as 1ng due to high resolution of the gel electrophoresis and strong specificity and high sensitivity of the immunoassay. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.

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Western Blot KEY Concepts

Western blotting principle usually involves two major processes, namely, SDS-polyacrylamide gel electrophoresis and protein blotting and testing.

Electrophoresis separation describes a phenomenon that charged particles move towards opposite electrode under the influence of electric field. It is used to separate proteins according to their electrophoretic mobility which depends on charge, molecule size and structure of the proteins. Polyacrylamide gel (PAG) is a three-dimensional mesh networks polymer composed of acrylamide and a cross-linker (methylene bisacrylamide) under the catalyzation of ammonium persulfate. PAG is a versatile supporting matrix due to its stable hydrophily and little adsorption and electroosmosis effect provided by its neutrally charged nature. (It possesses several electrophoretically desirable features that make it a versatile medium. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and can be prepared with a wide range of average pore sizes).

Coats protein with negative charge: In the presence of SDS, electrophoretic mobility is mainly based on molecule weight instead of on charge and size of the proteins. SDS is an anionic detergent which could break hydrogen bond within and between molecules to unfold proteins and break up secondary and tertiary structures as denaturing agent and hydrotropy agent. Strong reducing agents such as mercaptoethanol and Dithiothreitol (DTT) could disrupt disulfide linkages between cysteine residues. SDS and reducing agents are applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. This new negative charge is significantly greater than the original charge of that protein.

The electrostatic repulsion that is created by binding of SDS causes proteins to unfold into a rod-like shape thereby eliminating differences in shape as a factor for separation in the gel. Minor axis of all rods, the SDS-protein subunit compound are nearly the same, about 1.8nm. And the length of major axis is in proportion to molecular weight of the protein subunit. Thus electrophoretic mobility of the SDS-protein subunit compound is based on molecular weight, eliminating the influence imposed by size and charge.

The sample to be analyzed is mixed with SDS. And the mixed samples are subsequently treated by related solution. Heating the samples to at least 60°C further promotes protein denaturation and depolymerization, helping SDS to bind and enabling the rod-shape formation and negative charge adherence. A bromophenol blue dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run. An appropriate amount of glycerol is added to increase density and accelerate the migration of sample solution.

A buffer system with different pH values is applied in gel electrophoresis process. A very widespread discontinuous buffer system is the tris-glycine or "Laemmli" system that stacks at a pH of 6.8 and resolves at a pH of ~8.3-9.0. A drawback of this system is that these pH values may promote disulfide bond formation between cysteine residues in the proteins because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH values, so the gels can be stored for long periods of time before use.

As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is Cl¯ ( high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl¯ of the gel buffer and the Gly¯ of the cathode buffer. Because of the voltage drop between the Cl- and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layer-stacking gel layer.
In resolving gel layer, proteins with more negative charges per unit migrate faster than those with less negative charges per unit. That is, proteins with small molecular weight migrate faster than proteins with large molecular weight. The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occur continuously in the gradient gel, for every protein at a different position.

Choosing The Right Gel

How to choose the gel percentage, SDS-PAGE gel percentage calculator, the relationship between gel percentage and pore size

How to choose gel percentage based on protein size

Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel. Typically resolving gels are made in 5%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer and ladders will be placed) is inserted. The percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample. The smaller the known weight, the higher the percentage that should be used. Changes on the buffer system of the gel can help to further resolve proteins of very small sizes

Check the table below for common protein sizes and their recommended gel percentages

Range of molecular weight (KD) Concentration of gel (%)
<10 15
10 - 30 12
30 - 100 10
100 - 500 8
> 500 5


Five steps are involved in western blotting procedure and detection assay, namely, transfer, blocking, primary antibody incubation, secondary antibody incubation and protein detection, and western blotting analysis.

  • Transfer

    Proteins are moved from within the gel onto a membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF). Without pre-activation, proteins combine with nitrocellulose membrane based on hydrophobic interaction, thereby having slight effect on protein activities. Besides, nitrocellulose membrane produces little non-specific staining. It is cheap and ease to use. However, it is easy to erase small molecular proteins while washing. It is fragile and has poor toughness. With high affinity, the PVDF membrane needs to be sunk in methanol before use to activate positive charge groups on the membrane, promoting combination with negative charged proteins. Specific NC membrane with different pores should be applied according to the molecular weight of transferred proteins due to the smaller the pore of membrane the tighter the combination between membrane and small molecular weight proteins. NC membranes of 0.45 µm and of 0.2 µm are used most. The size of 0.45 µm should be applied for proteins with molecular weight over 20KD while the size of 0.2 µm will be chosen for those below 20KD. PVDF membrane is best for the detection of small molecular weight proteins due to its higher sensitivity, resolution as well as affinity than normal membrane.

    Transfer methods that are used most for proteins are semi-dry transfer and wet transfer. Semi-dry transfer describes the method that Gel-Membrane-Filter sandwich is placed between filters loaded with transfer buffer. The transfer process is based on current conduction produced by the transfer buffer. Semi-dry transfer takes little time with high efficiency as electric current works directly on membrane and gel. While applying wet transfer, the Gel-Membrane-Filter sandwich is placed in the transfer tank, suspending in transfer buffer vertically. Proteins transfer from the gel to the membrane under the control of high intensity electric field produced by electrode plate paralleled to the sandwich. While prolonging time to an appropriate extend, proteins could be transferred more effectively. Proteins within several gels could be transferred.

  • Blocking

    In a western blot, it is important to block the unreacted sites on the membrane to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay using inert protein or nonionic detergent. Blocking buffers should block all unreacted sites. And Blocking buffers should not replace target protein on the membrane, not bind epitope on the target protein and not cross react with antibody or detection reagents. The most typical blockers are BSA, nonfat dry milk, casein, gelatin and Tween-20. TBS and/or PBS are the most commonly used buffers.

    Inertia protein BSA, nonfat dry milk, casein, gelatin or nonionic detergent Tween-20 reduce nonspecific binding by blocking unreacted sites. Retaining protein structure, Tween-20 can reduce breakup to original interaction among proteins while is used for protein emulsification.

    1. Nonfat dry milk is the most economic choice
    2. Avoid using nonfat dry milk as a blocking reagent for blots with biotin conjugated antibody because milk contains variable amounts of glycoprotein and biotin.
    3. BSA is appropriate for blots with phosphorylated protein as target. Phosphatase contained in nonfat dry milk leads to dephosphorylation of phosphorylated protein on the membrane while phosphoryltion specific antibody is used to identify phosphorylated protein. And nonfat dry milk is improper for blots which rely on alkaline phosphatase system.
    4. Avoid adding NaN3 into blocking reagent for blots that base on HRP system because NaN3 is enabled to inactivate HRP.
    5. Casein is recommended for blots with alkaline phosphatase conjugated secondary antibody. TBS buffer instead of PBS buffer should be chosen because PBS interferes alkaline phosphatase.
  • Primary Antibody incubation

    After blocking, primary antibody specific to target protein is incubated with the membrane. And the primary antibody binds to target protein on the membrane.

    In western blot, primary antibody should be validated before use. The choice of a primary antibody depends on the antigen to be detected. Both polyclonal and monoclonal antibodies work well for western blot. Monoclonal antibodies recognize single specific antigenic epitope. Thus, they have higher specificity resulting in lower background. Blot results will be influenced if the target epitope is destroyed. Polyclonal antibodies recognize more epitopes and they often have higher affinity. Blot results will be stable even though a few epitopes are destroyed.

  • Secondary antibody incubation

    After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to a specific enzyme-conjugated secondary antibody. And the secondary antibody binds to the primary antibody which has reacted with the target protein.

    The most popular secondary antibodies are anti-mouse and anti-rabbit immune globulin since the host species for primary antibodies are mainly mouse and rabbit. Goat is used widely to raise anti-mouse and anti-rabbit polyclonal antibodies. Thus, goat anti-mouse and goat anti-rabbit immune globulin are the most commonly used secondary antibodies. The choice of secondary antibody depends upon the species of animal in which the primary antibody was raised. For example, if the primary antibody is a mouse monoclonal antibody, the secondary antibody must be an anti-mouse antibody. If the primary antibody is a rabbit polyclonal antibody, the secondary antibody must be an anti-rabbit antibody.

    Protein detection (color development) and analysis of Protein detection (color development)

    A substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance, namely, visible protein bands. The target protein levels in cells or tissues are evaluated through densitometry and the location of the visible protein bands.

    Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes that are used extensively. Functioned by Alkaline phosphatase (AP) catalyzation, a colorless substrate BCIP will be converted to a blue product. In the presence of H2O2, 3-amino-9- ethyl carbazole and 4-chlorine naphthol will be oxidized into brown substance and blue products respectively under the catalyzation of HRP. Enhanced chemiluminescence is another method that employs HPR detection. Using HRP as the enzyme label, luminescent substance luminol will be oxidized by H2O2 and will luminesce. Moreover, enhancers in this substrate will enable a 1000-fold increase in light intensity. HRP will be detected when the blot is sensitized on photographic film.

  • Western Blotting Analysis

    After color development, the pattern of the separated proteins is imprinted onto the film or captured by Western Blot gel imager. By comparing the band position to the protein ladder, one can estimate the size of the protein.

Would you like a free Western Blot Guide?

This guide will teach you everything you need to become a Western Blot (WB) expert, including a comprehensive principle overview, insightful troubleshooting tips, and more.

Recommended reagents

The reagents you will need for each step are listed below.

Western blot control design

5 types of common controls

No controls, no science

Proper control design is essential to western blot. It will guarantee accurate and specific test result by identifying various problems quickly and precisely. There are 5 common types of controls seen in Western blot experiment design.

  1. Positive control: A lysate from a cell line or tissue sample known to express the protein you are detecting. Positive control is designed to verify working efficiency of the antibodies.
  2. Negative control: A lysate from a cell line or tissue sample known not to express the protein you are detecting. Negative control is to check antibody specificity. Nonspecific binding and false positive result will be identified.
  3. Secondary antibody control (No primary antibody control): The primary antibody is not added to the membrane. Only secondary antibody is added. This is to check secondary antibody specificity. Nonspecific binding and false positive result caused by secondary antibody will be indicated.
  4. Blank control: Both primary and secondary antibody are not added to membrane. This is to check membrane nature and blocking effect.
  5. Loading control: Loading control is used to check sample quality and the performance of secondary antibody system.

More about loading controls

Loading controls are antibodies to "house-keeping proteins", or proteins that are expressed at equivalent levels in almost all tissues and cells.

Loading controls are required to check that the lanes in your gel have been evenly loaded with sample, especially when a comparison must be made between the expression levels of a protein in different samples. They are also useful to check for even transfer from the gel to the membrane across the whole gel. Where even loading or transfer have not occurred, the loading control bands can be used to quantify the protein amounts in each lane. For publication-quality work, use of a loading control is absolutely essential

Loading Control Molecular Weight (KD) Sample Type
Beta-actin 43KD Whole Cytoplasmic
GAPDH 30 - 40KD Whole Cytoplasmic
Tubulin 55KD Whole Cytoplasmic
VCDA1/Porin 43KD Mitochondrial
COXIV 16KD Mitochondrial
Lamin B1 16KD Nuclear (Not suitable for samples where the nuclear envelope is removed.)
TBP 38KD Nuclear (Not suitable for samples where the nuclear envelope is removed.)

More western blot resources

sample preparation, protocol, troubleshooting and more.

WB Sample Preparation

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WB Protocol

A step-wise guide of optimized WB protocol. Best choice for setting up a new SOP for your lab or for educating new lab members.

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WB Troubleshooting

Something wrong with your blot? No worries, check out the comprehensive troubleshooting guide to see if your issue is already covered.

WB troubleshooting

Popular Antibodies

Here are the 300 most popular antibodies.

acetylcholinesterase antibodyADAM10 antibodyADAMTS13 antibodyadiponectin antibody
AGO2 antibodyAHR antibodyALK antibodyalkaline phosphatase antibody
AMH antibodyANG antibodyannexin a1 antibodyAPOE antibody
AQP1 antibodyaquaporin 4 antibodyARC antibodyATF4 antibody
ATF6 antibodyATM antibodyATRX antibodyBAX antibody
bcl xl antibodyBCR antibodybeclin 1 antibodyBRCA1 antibody
CAD antibodycalreticulin antibodycaspase 1 antibodycaspase 8 antibody
catalase antibodycathepsin b antibodyCcl2 antibodyCCR4 antibody
CCR5 antibodycd11b antibodycd11c antibodyCd14 antibody
cd16 antibodyCD163 antibodyCD19 antibodycd2 antibody
cd20 antibodyCD200 antibodyCD24 antibodyCD27 antibody
cd30 antibodyCD33 antibodyCD34 antibodyCD36 antibody
Cd40 antibodyCD44 antibodycd45 antibodyCD47 antibody
CD63 antibodyCD68 antibodycd8 antibodyCD80 antibody
CD81 antibodyCD86 antibodyCd9 antibodyCDK2 antibody
CDK4 antibodyceruloplasmin antibodyCHAT antibodyclaudin 5 antibody
collagen i antibodycollagen ii antibodycreb antibodyCrp antibody
csf antibodyCSF1R antibodyCtla4 antibodyCXCL10 antibody
CXCR3 antibodyCXCR4 antibodycyclin antibodycytokeratin 5 antibody
desmoglein 3 antibodyDKK1 antibodyDLL4 antibodyDNMT1 antibody
doublecortin antibodyEEA1 antibodyEGFR antibodyenos antibody
EPCAM antibodyERBB2 antibodyerythropoietin antibodyEZH2 antibody
fak antibodyFAP antibodyFAS antibodyFGF21 antibody
Fgf23 antibodyFGFR1 antibodyFLT3 antibodyFOS antibody
FOXO1 antibodyFOXP3 antibodyGAL antibodyGAPDH antibody
GATA3 antibodyGDF15 antibodyglut4 antibodygp100 antibody
GPI antibodyGPX4 antibodygrp78 antibodyHGF antibody
HIF1A antibodyHLA-A antibodyhsp90 antibodyhuntingtin antibody
iba1 antibodyICAM1 antibodyIcos antibodyIDS antibody
IFNAR1 antibodyIgf1r antibodyIl10 antibodyIL13 antibody
IL15 antibodyIl17a antibodyIL18 antibodyIL1B antibody
IL2 antibodyIL33 antibodyIL5 antibodyIL6R antibody
IL8 antibodyinos antibodyIRF3 antibodyislet 1 antibody
JAK2 antibodyjnk antibodyKEAP1 antibodyKLF4 antibody
L1CAM antibodylactoferrin antibodylamin a antibodyLAMP1 antibody
LAT antibodyleptin antibodyLif antibodyLOX antibody
lysozyme antibodyMAG antibodyMAX antibodyMBP antibody
MDM2 antibodyMERTK antibodymesothelin antibodyMICA antibody
MIF antibodyMLH1 antibodyMMP2 antibodyMMP9 antibody
MOG antibodyMTOR antibodyMUC2 antibodymyeloperoxidase antibody
NANOG antibodynephrin antibodynestin antibodyNGF antibody
NLRP3 antibodyNOTCH1 antibodyNOX4 antibodyNPY antibody
NRP1 antibodyoccludin antibodyosteocalcin antibodyosteopontin antibody
p300 antibodyp63 antibodyparkin antibodyparp antibody
PAX6 antibodyPAX8 antibodyPCSK9 antibodypd l1 antibody
PDGFRA antibodyPDGFRB antibodyperilipin antibodyperk antibody
Pf4 antibodypgp9.5 antibodyPML antibodyppar gamma antibody
PROX1 antibodyPTEN antibodyrab7 antibodyRAC1 antibody
RAD51 antibodyrea antibodyRET antibodyrip3 antibody
RIPK1 antibodyRUNX2 antibodySHH antibodySIRT1 antibody
SMAD2 antibodysmad3 antibodySNAP25 antibodySOD1 antibody
SOD2 antibodysomatostatin antibodySOX10 antibodySOX2 antibody
SOX9 antibodySP1 antibodySPR antibodySRC antibody
STAT3 antibodySTAT6 antibodysurvivin antibodySYK antibody
TAZ antibodyTBK1 antibodytdt antibodyTERT antibody
thrombin antibodyThy1 antibodytim 3 antibodyTLR2 antibody
TLR3 antibodyTLR4 antibodyTnf antibodytopoisomerase i antibody
transferrin antibodytrkb antibodyTSG101 antibodyTSHR antibody
TSLP antibodyubiquitin antibodyVDAC1 antibodyvegf antibody
VWF antibodyXBP1 antibodyZEB2 antibody

Western Blotting FAQs

A western blot is a laboratory method used to detect specific protein molecules from  among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type.

Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein.

Western Blotting (also called immunoblotting) is a technique used for analysis of individual proteins in a protein mixture (e.g. a cell lysate). The proteins on this immunoblot are then accessible for antibody binding for detection. Antibodies are used to detect target proteins on the western blot (immunoblot).

Primary antibodies directly bind to the protein of interest, but unless they are directly conjugated to a dye or an enzyme, a secondary antibody is needed for detection. Conjugated secondary antibodies are used to detect the primary antibody.

Western Blotting is the most common method of testing to confirm positive results from ELISA tests.One advantage of Western Blotting is that it's less likely to give false positive results as it can effectively distinguish between different antibodies.

Western blot is a reliable quantitative method only if sample properties and integrity, antibody specificity to the target protein, and loading protocols are considered. With careful attention to details, you can avoid common mistakes and avoid misinterpreting Western blot data.