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What is western blotting, the principle behind western blotting, how western blot works step-by-step, and a detailed WB protocolBrowse Boster Featured Products
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 blotting principle usually involves two major processes, namely, SDS-polyacrylamide gel electrophoresis and protein blotting and testing.
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.
How to choose the gel percentage, SDS-PAGE gel percentage calculator, the relationship between gel percentage and pore 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
|Range of molecular weight (KD)||Concentration of gel (%)|
|10 - 30||12|
|30 - 100||10|
|100 - 500||8|
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.
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.
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.
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.
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.
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.
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.
The reagents you will need for each step are listed below.
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.
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|
|GAPDH||30 - 40KD||Whole Cytoplasmic|
|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.)|
sample preparation, protocol, troubleshooting and more.
The choice of protein extraction method is crucial. It ultimately makes the difference between a blank blot and a beautiful one.WB sample preparation
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.WB protocol
Something wrong with your blot? No worries, check out the comprehensive troubleshooting guide to see if your issue is already covered.WB troubleshooting
Here are the 300 most popular antibodies.
|14 3 3 antibody||Actin antibody||Ada antibody||Afp antibody|
|Akt1 antibody||alix antibody||APP antibody||B2M antibody|
|bcl 2 antibody||caspase 3 antibody||CAV1 antibody||CCR2 antibody|
|CD22 antibody||cd3 antibody||CD4 antibody||CD46 antibody|
|CD5 antibody||CDK1 antibody||CEACAM1 antibody||CEACAM5 antibody|
|collagen iii antibody||collagen iv antibody||cytochrome c antibody||desmin antibody|
|erk1 antibody||fibronectin antibody||GFAP antibody||glut1 antibody|
|gm130 antibody||HMGB1 antibody||hsp70 antibody||IDH1 antibody|
|ifn gamma antibody||Il6 antibody||IRS1 antibody||laminin antibody|
|MAP2 antibody||MECP2 antibody||MUC1||MYC antibody|
|myoglobin antibody||ncam antibody||p19 antibody||p53 antibody|
|p62 antibody||paxillin antibody||PCNA antibody||PLK1 antibody|
|RPS6 antibody||S100B antibody||SMAD4 antibody||STAT1 antibody|
|synaptophysin antibody||tau antibody||TFAM antibody||tgfb antibody|
|tyrosine hydroxylase antibody||UPF1 antibody||vimentin antibody||vinculin antibody|
|acetylcholinesterase antibody||ADAM10 antibody||ADAMTS13 antibody||adiponectin antibody|
|AGO2 antibody||AHR antibody||ALK antibody||alkaline phosphatase antibody|
|AMH antibody||ANG antibody||annexin a1 antibody||APOE antibody|
|AQP1 antibody||aquaporin 4 antibody||ARC antibody||ATF4 antibody|
|ATF6 antibody||ATM antibody||ATRX antibody||BAX antibody|
|bcl xl antibody||BCR antibody||beclin 1 antibody||BRCA1 antibody|
|CAD antibody||calreticulin antibody||caspase 1 antibody||caspase 8 antibody|
|catalase antibody||cathepsin b antibody||Ccl2 antibody||CCR4 antibody|
|CCR5 antibody||cd11b antibody||cd11c antibody||Cd14 antibody|
|cd16 antibody||CD163 antibody||CD19 antibody||cd2 antibody|
|cd20 antibody||CD200 antibody||CD24 antibody||CD27 antibody|
|cd30 antibody||CD33 antibody||CD34 antibody||CD36 antibody|
|Cd40 antibody||CD44 antibody||cd45 antibody||CD47 antibody|
|CD63 antibody||CD68 antibody||cd8 antibody||CD80 antibody|
|CD81 antibody||CD86 antibody||Cd9 antibody||CDK2 antibody|
|CDK4 antibody||ceruloplasmin antibody||CHAT antibody||claudin 5 antibody|
|collagen i antibody||collagen ii antibody||creb antibody||Crp antibody|
|csf antibody||CSF1R antibody||Ctla4 antibody||CXCL10 antibody|
|CXCR3 antibody||CXCR4 antibody||cyclin antibody||cytokeratin 5 antibody|
|desmoglein 3 antibody||DKK1 antibody||DLL4 antibody||DNMT1 antibody|
|doublecortin antibody||EEA1 antibody||EGFR antibody||enos antibody|
|EPCAM antibody||ERBB2 antibody||erythropoietin antibody||EZH2 antibody|
|fak antibody||FAP antibody||FAS antibody||FGF21 antibody|
|Fgf23 antibody||FGFR1 antibody||FLT3 antibody||FOS antibody|
|FOXO1 antibody||FOXP3 antibody||GAL antibody||GAPDH antibody|
|GATA3 antibody||GDF15 antibody||glut4 antibody||gp100 antibody|
|GPI antibody||GPX4 antibody||grp78 antibody||HGF antibody|
|HIF1A antibody||HLA-A antibody||hsp90 antibody||huntingtin antibody|
|iba1 antibody||ICAM1 antibody||Icos antibody||IDS antibody|
|IFNAR1 antibody||Igf1r antibody||Il10 antibody||IL13 antibody|
|IL15 antibody||Il17a antibody||IL18 antibody||IL1B antibody|
|IL2 antibody||IL33 antibody||IL5 antibody||IL6R antibody|
|IL8 antibody||inos antibody||IRF3 antibody||islet 1 antibody|
|JAK2 antibody||jnk antibody||KEAP1 antibody||KLF4 antibody|
|L1CAM antibody||lactoferrin antibody||lamin a antibody||LAMP1 antibody|
|LAT antibody||leptin antibody||Lif antibody||LOX antibody|
|lysozyme antibody||MAG antibody||MAX antibody||MBP antibody|
|MDM2 antibody||MERTK antibody||mesothelin antibody||MICA antibody|
|MIF antibody||MLH1 antibody||MMP2 antibody||MMP9 antibody|
|MOG antibody||MTOR antibody||MUC2 antibody||myeloperoxidase antibody|
|NANOG antibody||nephrin antibody||nestin antibody||NGF antibody|
|NLRP3 antibody||NOTCH1 antibody||NOX4 antibody||NPY antibody|
|NRP1 antibody||occludin antibody||osteocalcin antibody||osteopontin antibody|
|p300 antibody||p63 antibody||parkin antibody||parp antibody|
|PAX6 antibody||PAX8 antibody||PCSK9 antibody||pd l1 antibody|
|PDGFRA antibody||PDGFRB antibody||perilipin antibody||perk antibody|
|Pf4 antibody||pgp9.5 antibody||PML antibody||ppar gamma antibody|
|PROX1 antibody||PTEN antibody||rab7 antibody||RAC1 antibody|
|RAD51 antibody||rea antibody||RET antibody||rip3 antibody|
|RIPK1 antibody||RUNX2 antibody||SHH antibody||SIRT1 antibody|
|SMAD2 antibody||smad3 antibody||SNAP25 antibody||SOD1 antibody|
|SOD2 antibody||somatostatin antibody||SOX10 antibody||SOX2 antibody|
|SOX9 antibody||SP1 antibody||SPR antibody||SRC antibody|
|STAT3 antibody||STAT6 antibody||survivin antibody||SYK antibody|
|TAZ antibody||TBK1 antibody||tdt antibody||TERT antibody|
|thrombin antibody||Thy1 antibody||tim 3 antibody||TLR2 antibody|
|TLR3 antibody||TLR4 antibody||Tnf antibody||topoisomerase i antibody|
|transferrin antibody||trkb antibody||TSG101 antibody||TSHR antibody|
|TSLP antibody||ubiquitin antibody||VDAC1 antibody||vegf antibody|
|VWF antibody||XBP1 antibody||ZEB2 antibody|