Introduction

Many biological and medical diagnostics laboratories employ Western blotting (a.k.a. immunoblotting) to detect proteins from cell extracts, blood serum or saliva sputum samples. Western blotting is considered a mature technology, but remains extensively used by researchers in the biotechnology, pharmaceutical and diagnostics industries. Customers continue to demand improvements in ease-of-use, speed, and sensitivity, as well as, a better ability to quantify results. 

Western blot analysis can detect one protein in a mixture of any number of proteins while providing you with information about the molecular weight of the protein. This method, however, is dependent on the use of a high-quality antibody directed against a desired protein. This antibody is used as a probe to detect the protein of interest.

Western Blot Analysis Services

· Extraction of total proteins from tissues/cells provided by Leading Biology or customers

· Constructions of Western blots using total proteins

· Immunostaining blots with antibodies provided by customers using ultrasensitive chemiluminescent ECL detection

· Stripping antibodies from blots 

· Reimmunostain blots with any house-keeping gene antibodies

· Scanning protein bands on X-ray films

· Quantitative analysis of immunostaining data

· Sending high quality digital images of X-ray films on Microsoft PowerPoint

Western Blotting Workflow

The most commonly used protein blotting technique, western blotting (immunoblotting), was developed to probe for proteins that were inaccessible to antibodies while in polyacrylamide gels. Western blotting refers specifically to the immunological detection of proteins that have been separated by gel electrophoresis and transferred onto a membrane. Since the development of immunoblotting techniques, other probing and detection techniques have been developed for functional protein characterization.

The western blotting workflow involves two phases: protein transfer to a membrane and detection of the membrane-immobilized protein.

Protein Transfer to a Membrane

The first phase of western blotting is the transfer step, which consists of moving the proteins from a solution or gel matrix to a synthetic membrane support where it is bound, forming the blot. Proteins can be transferred to membranes using a number of methods, but the most common are electrophoretic transfer (electroblotting) and microfiltration (dot blotting). 

In general, electrophoretic transfer is used to transfer proteins following electrophoretic separation by native or SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and microfiltration is used to transfer proteins that are in solution. Capillary blotting methods, such as those used for nucleic acid transfer, are seldom used to transfer proteins from gels.

Protein Detection on the Membrane

The second phase, protein detection, entails probing the membrane with either a protein stain or antibodies specific to the protein of interest, and subsequent visualization of the labeled proteins. Western blot detection involves a number of steps, including selection of the appropriate protein detection method, blotting buffers and reagents, and gel and blot imaging equipment.

The protein blotting workflow involves the selection of the appropriate method, apparatus, membrane, buffer, and transfer conditions. Once proteins are immobilized on a membrane, they are available for visualization, detection, and analysis.

Get started

Please contact us with your Western Blot services requests at info@leadingbiology.com and we will reply with a detailed quote as soon as possible. This process usually takes between 24 and 48 hours and the quote will include an estimated price as well as the time required to complete the project. All inquiries and subsequent projects are handled in strict confidence and will be backed by a confidentiality agreement if desired.