Antibodies for Western Blot
Primary Antibodies
Primary Antibodies
Bio-Techne® offers a large portfolio of primary antibodies validated for Western blot experiments.
Secondary Antibodies
Secondary Antibodies
Bio-Techne offers enzyme- and fluorochrome-conjugated secondary antibodies for use in Western blot experiments.
Epitope Tag Antibodies
Epitope Tag Antibodies
Bio-Techne offers antibodies for the most commonly used epitope tags for the detection of tagged proteins with Western blot.
Novus Biologicals™ Western Blot Tools
Novus Biologicals offers reagents for your entire Western blot workflow including over 65,000 Western blot-validated antibodies, HRP-conjugated secondary antibodies, detection reagents, and controls such as blocking peptides, control lysates, and over-expression lysates.
Western Blot Imaging and Analysis
FluorChem™ Imagers
FluorChem™ Imagers
Bio-Techne’s FluorChem imagers are all-in-one solutions for high-quality multimodal imaging of Western blot membranes and electrophoresis gels.
AlphaImager® Systems
AlphaImager® Systems
Bio-Techne’s AlphaImager systems are digital gel imagers and documentation systems for colorimetric and fluorescent research applications.
AlphaView® Software
AlphaView® Software
Bio-Techne’s AlphaView software allows for the easy capture, analysis, and annotation of membrane and gel images. Optimized for use with our FluorChem and AlphaImager systems.
Simple Western™ Systems
Jess™
Jess™
Bio-Techne’s Jess is an automated capillary-based Western blotting system. Its capabilities include chemiluminescent immunodetection, two-color fluorescence, and protein normalization.
Wes™
Wes™
The predecessor to Jess, Bio-Techne’s automated, capillary-based Western blotting system, Wes, separates and analyzes proteins by chemiluminescence.
Sally Sue™
Sally Sue™
Bio-Techne's Sally Sue lets you separate and analyze proteins by size in an automated fashion. Only 5 µL of sample is needed, and up to 96 samples can be run in one experiment.
Peggy Sue™
Peggy Sue™
Bio-Techne’s Peggy Sue system lets you separate and analyze proteins by either size or charge in an automated fashion. Only 5 µL of sample is needed, and up to 96 samples can be run in one experiment.
NanoPro™ 1000
NanoPro™ 1000
The NanoPro 1000 is Bio-Techne’s automated, capillary-based nano-immunoassay platform that uses isoelectric focusing to characterize protein post-translational modifications.
Compass Software
Compass Software
Analyze Simple Western size or charge assay data with Bio-Techne’s Compass software. Equipped with features that satisfy the 21 CFR Part 11 data security requirements.
Perform Western Blotting in Single Cells
Our single-cell Western platform measures protein expression in ~1,000 single cells in a single run.
Related Western Blot Resources
Western Blot Handbook
Western Blot Handbook
This step-by-step Western blot guide from Novus Biologicals is a great introduction to Western blot. It provides detailed information for understanding, performing, and troubleshooting a Western blot protocol.
Western Blot Troubleshooting Resource
Western Blot Troubleshooting Resource
A comprehensive, online resource for troubleshooting the most common problems of Western blot including no signal, multiple bands, and high background.
Development of Validated Monoclonal Antibodies
Development of Validated Monoclonal Antibodies
This white paper describes our validation process, including antigen design, feasibility testing, specificity and sensitivity testing, and quality control and release decisions, for our monoclonal antibodies.
Background Information
Western blot is a laboratory method that uses antibodies to identify individual proteins in cell or tissue lysates. Due to the specific nature of antibody binding, Western blot analysis can be used to detect and quantify a single protein within a mixture of thousands of different proteins. In Western blot, samples are loaded onto a polyacrylamide gel and separated based on their molecular weight by SDS-PAGE electrophoresis. Polyacrylamide gels that are used consist of two sections: a stacking gel and a separating gel. The stacking gel contains less acrylamide and has a lower pH. This environment allows the proteins in the sample to form highly defined, sharp bands. The proteins pass from the stacking gel into the separating gel, which is more basic and has a higher gel concentration. This causes the proteins to separate by size, with the smaller proteins traveling faster through the gel. The proteins are then transferred to a membrane by application of an electrical current. The membrane can be incubated with primary antibodies specific for target protein(s) of interest. Secondary antibodies bound to enzymes or fluorochromes are applied to the membrane to visualize the protein/antibody complex. A band will appear at approximately its molecular weight, as marked by the molecular weight ladder.
What is SDS-PAGE?
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is an electrophoresis method commonly used to separate proteins based by size. SDS is an anionic detergent, a detergent that has a negative charge, that binds to proteins and disrupts the non-covalent forces that are responsible for their three-dimensional structure, thus denaturing the proteins. SDS also gives the proteins a uniform net negative charge that is relative to its size. This causes the denatured proteins to migrate through the gel during electrophoresis solely based on its size. People typically do SDS-PAGE prior to Western blotting. SDS-PAGE separates the proteins while Western blotting involves the transfer of proteins from the gel to a membrane, and confirmation of the presence/absence/expression level of the proteins using an antibody.
What is native PAGE and why might it be used?
Native or non-denaturing gels do not use SDS. Nor is a reducing agent, such as DTT or beta-mercaptoethanol, added to the sample loading buffer. This means that the proteins maintain their native structure and charge. As a result, they will migrate through the gel during electrophoresis according to both their mass and their charge. Native gels are used to determine the aggregation state of a protein, study protein complexes, and isolate enzymes.
How much sample should be loaded for a Western blot?
The amount of protein that should be loaded into the loading gel for a Western blot depends a several factors including the expression level of the protein and the sample being probed. As a rule of thumb, 20-30 µg of total protein is loaded per well for cell and nuclear lysates and membrane samples. Ten to 100 ng of protein is typically loaded if probing for a purified protein. The appropriate protein amount to load, though, should be determined prior to starting a Western blot experiment.
What types of controls are needed for a Western blot?
Proper controls for Western blotting are important for determining the source of problems and for validating results. There are several controls that need to be used for a proper Western blot experiment.
1. Positive Control Lysates – Positive control lysates are from cell lines or tissue samples that are known to express the protein of interest. This control will yield a positive band on the Western blot. This control is important as it ensures there were no issues in the Western blot protocols and that any negative results are due to a lack of protein in the sample and not to procedural problems.
2. Negative Control Lysates – Negative control lysates are from samples known to not express the target protein. This control will yield no band on the Western blot. This control is important for determining non-specific binding of the antibodies.
3. Positive Endogenous Control Lysates - Positive endogenous control lysates are from samples that are known to express the target of interest. This control should be used when testing a sample of recombinant protein, such as a tagged protein. Folding of a recombinant protein may be different than the native protein, and misfolding may prevent the antibody from accessing the epitope. This control will let the researcher know that a negative result is due to the epitope being blocked as opposed to the protocol not working.
4. Loading Control – Loading controls are housekeeping proteins, which are proteins that are expressed at equivalent levels in almost all tissues and cells. This control ensures that differences in protein expression between wells isn’t due to loading or protein transfer errors. Loading controls are required for the semi-quantification of protein levels between wells.
What is the Difference Between Simple Western and Western Blot?
Simple Western assays are automated Western blot. This platform automates all the steps involved in electrophoresis and Western blotting, from protein loading and separation, immunoprobing, washing, detection and quantitative analysis of data. View the Simple Western FAQs page on the Novus Biologicals website for more information on Simple Western.
