Western blot introduction

What is Western Blot

Chemical coloration detection for western blotWhat's western blot test? Western blot test is a technique used to detect specific proteins from samples such as cell extracts or tissue homogenates, also analyze recombinant proteins synthesized in vitro. Western blot can also be used to identify a target protein on the basis of the special affinity between a certain antigen and its relative antibody. Western blot generally contains three main steps, SDS-PAGE firstly, then blot of samples, and finally immunology test. Qualitative and semi-quantitative data can be acquired about the target protein through western blot. If both qualitative and quantitative data is wanted, another technique ELISA is suggested. Learn more about the traits comparison between western blot and other methods like ELISA, IHC/ICC, and IFA (Immunofluorescence assay). Besides, about western blot test, it's necessary to mention that the name "Western blot " is a play on the name of southern blot test, which is named after its inventor Edwin Southern. In the field of protein, western blot is widely used for the test of protein expression level.

Western Blot Antibody

Rabbit Monoclonal Antibody for Western Blot

Advantages of Rabbit Monoclonal Antibody (RabMAb) 

Monoclonal antibodies are traditionally produced by hybridoma - myeloma cells fused with the spleen cells, from a mouse which has acquired immunization of the specific antigen. It is cost-effective and easy to operate to use a mouse as a host to produce monoclonal antibodies. However, researchers have been confronted with a problem of mouse monoclonal antibodies - limitations. Firstly, many target proteins are highly conservative between mice and humans, and therefore, those proteins may be recognized as self-antigens by a mouse host, making the antigens less immunogenic than expected. Secondly, compared with rabbit monoclonal antibodies, mouse monoclonal antibodies have lower affinities, and thus have a weak attraction to bind to the target proteins. 

Polyclonal antibody

Polyclonal antibodies are antibodies that are derived from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope. The immunization of a suitable mammal, such as a mouse, rabbit or goat produce these antibodies.When an antigen is injected into the mammal, it induces the B-lymphocytes to produce IgG immunoglobulins that is specific for the antigen.

Secondary Antibody Overview

A secondary antibody which binds to a primary antibodyA secondary antibody is an antibody that binds to a primary antibody or antibody fragments. Secondary antibodies which are marked as probes are useful for the detection, purification or cell sorting applications. Secondary antibodies may be monoclonal or polyclonal. They are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions. Specific secondary antibodies are usually used to work in specific laboratory applications. Frequently, any one of several secondary antibodies has a particular application adequately. They are chosen according to the source of the primary antibody, the class of the primary antibody (e.g., IgM or IgG), and the kind of label which is preferred. The optimal secondary antibody is normally identified through trial and error. Secondary antibodies have been very useful for many biochemical assays, including immunocytochemistry, ELISA, western blot, immunohistochemistry, and so on.

Western Blot Loading Control Antibody Background

Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample you are examining. This ensures constant expression levels. Thus "housekeeping genes" are frequently chosen for use as loading controls. It is also important that the protein chosen as a loading control has a different molecular weight than the protein of interest so that the bands are distinct and expression levels quantifiable. Popular loading control detection antibodies include anti-β-Actin monoclonal or polyclonal antibodies, anti-COX-4, anti-GAPDH, anti-Tubulin and anti-VDAC/Porin antibodies.

Western Blot Isotype Control Antibody Background

Isotype Control IgG is essential for ELISA, Western Blot (WB), Immunohistochemistry (IHC) and Immunoprecipitation (IP) experiments. It's purpose is to estimate that the proteins stained in the experiment result are due to the specific interaction with the antibody. Some people use specific primary antibody alone. This does not consider of those proteins that may bind to regions of the immunoglobulin distinct from the specific antigen binding sites. This Non-Specific binding is due to Fc receptor binding or other protein-protein interactions.The best way is to use isotype-matched control for the experiment. The IgG isotyping control should have the same immunoglobulin subtype and be used at the same concentration as the specific detection antibody. Sino Biological has a very good selection of control IgGs and the quality is excellent. 

Western Blot Tag Antibody Background

Tag antibodies make it easy to detect expression of proteins with specific tags, Myc, GST, GFP, His, HA, etc. Besides, some tag antibodies can be used in purification of tag fusion proteins. Sino Biological offers quality tag antibodies for your research needs with affordable price, 30-80% cost saving compared with currently leading brands; and big discount for bulk order.

Tag antibodies are widely used in various applications, either in detection of protein expression, or cellular localization, and protein purification. Sino Biological provides high quality tag antibodies validated in many immunoassays, western blot, ELISA, etc.

Antibody
Antibody
Western blotting / WB Antibody
Western blotting / WB Antibody Features
WB Products Center
Western Blot / WB Technique Center+
- Western blot introduction
What is Western Blot
Western Blot for Diagnosis
Western Blot Applications for Research
- Western Blot / WB tips
Sinobiological Western blot general tips
Western Blotting: 10 Technical Tips for Success
- Western Blot / WB FAQ
1. Why are there multiple bands on my blot?
2. Why is there a weak signal or no signal at all?
3. Why is there high background and/or diminished signal with anti-phosphor tyrosine antibodies?
4. Why does my antibody activity diminish in Western Blot over time?
5. How do I overcome inadequate or poor transfer problems?
6. How do I avoid "splotchy" or uneven Western Blots?
7. What can I use as positive control lysates for sinobiological WB antibodies?
8. Too many bands on a Western blot
9. No Signal or Weak Signal
10. Nonspecific Bands
11. High Background
12. How much protein should I analyse?
13. What percentage acrylamide gel should I use to resolve the proteins?
14. What membrane should I use?
15. What blocking buffer should I use?
16. What dilution of primary antibody should I use?
- Western Blot / WB protocol
Far Western Blot
ECL Western Blot
HRP Western Blot
Wet Western Blot
Semi-dry Western Blot
Densitometry Western Blot
Fluorescent Western Blot
Chemiluminescence Western Blot
Membrane Protein Western Blot
Western Blot Buffer / Reagents
Western Blot Sample Preparation
Western Blot Gel Electrophoresis
Western Blot Transfer
Western Blot Blocking
Western Blot Antibody Incubation
Western Blot Detection
- Western Blot / WB trouble shooting
High Background
Speckled Background
Multiple Bands
No Bands
Low Signal
Other Problems