western blot protocols, troubleshooting, scientific resources and research articles for the research community and science students
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In western blotting (or immunoblotting), prior to protein immobilization on the PVDF or nitrocellulose membranes, sample proteins are separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) providing information about molecular weight and the potential existence of different isoforms of the proteins under study.
1. PubMed: comprehensive database - 15 million citations
2. NIH:the National Institutes of Health
3. UniProt: The world's most comprehensive catalogue of information on proteins
4. Western
Blot Procedure Western blot protocol overview
2. Air pockets are not compatible with the passage of electric current. Pressing the gel and membrane ‘sandwich’ together by gently using a Pasteur pipette as a rolling pin can reduce the occurrence of air pockets between the layers.For more western blot tips, see the western blot Troubleshooting section.
Ion
Channels - Protein Extraction and Western Blot
Excitable cells, such as cardiomyocytes and neurons depolarize and repolarize
in an ordered, temporal manner in a process known as the action potential.The
movement of ions is driven by the respective electrochemical gradients
for those ions.
The plasma membrane is impermeable to these ions and, in order for ions
to move down their respective electrochemical gradients, specific pores
(ion channels, pumps or exchangers) need to allow them to pass. The opening
and closing of ion channels located in cellular membranes allows for the
coordinated flux of ions across the phospholipid barrier.
A principal determinant of the excitable cell behavior is the protein expression levels of its complement of ion channels. Those who study cardiac pathologies (atrial arrhythmias, congestive heart failure, ..) often measure differences in ion channel expression and distribution in control and diseased hearts. The subcellular distribution of various proteins can be assessed using immunohistochemistry methods.
Western blotting provides a method for protein
expression level comparisons, however several factors need to be considered.
western blotting and ion channels continued
1.The first paper describing the use of electrophoresis to transfer proteins from a gel to a membrane. Towbin H., Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4
2.The classic paper by UK Laemmli - the most cited article in the journal's history. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680-5.
3. The first paper to use the term western blot. Burnette
WN. "Western blotting": electrophoretic transfer of proteins
from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose
and radiographic detection with antibody and radioiodinated protein A.
Anal Biochem. 1981 Apr;112(2):195-203
Dot blot methodology differs from traditional western blot techniques by spotting sample proteins onto the membrane (some protocols use a vacuum)-not by electrophoresis.
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Text Research Articles
Guennadi A Khoudoli , Iain M Porter , J Julian Blow and Jason R Swedlow
Keywords: Xenopus egg extract, chromatin, cell cycle, two-dimensional gel electrophoresis, proteomics, Taguchi method, optimisation
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a molecular biology protocol that allows for the separation of molecules (the sample proteins for western blotting). Proteins are electrophoretically separated based on weight and electrical properties as they migrate through a polyacrylamide gel matrix. Once separated, proteins can be electrophoretically transferred to a membrane for immunoblot assay.
Western immunoblots provide more specific results than the ELISA protocol by providing additional information regarding protein size and multiple bands. Western blot results help confirm if someone is HIV positive because some conditions (including lupus and lyme disease) may inaccurately yield a positive ELISA test result.
Western blot methods are used in HIV research as well. In a recent study by Suthon et al., it was reported that the use of an HIV vaccine induced an antibody response that complicated anti-HIV-1screening tests among vaccinees.The western blot test detected the vaccine induced antibody (click for full text article: Impact of HIV vaccination on laboratory diagnosis: case reports
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Avian influenza (bird flu) is a viral infection that can be readily transmitted among poultry and other birds. There are various strains of the type A influenza virus which cause illness. The highly pathogenic H5 and H7 subtypes-specifically the H5N1, H7N7 and H7N3 strains-account for most of the cases of human illness. The less pathogenic H9N2 avian influenza strain has also affected people.*
With growing concern about a possible avian flu pandemic, much research is being conducted with the aim of developing a vaccine. In their recent paper, Nwe et al describe the expression of hemagglutinin (HA1) from avian influenza H5N1 in monolayer or suspension culture insect cells. The authors used SDS-PAGE and immunoblot analysis of rHA1 protein expressed in a baculovirus/insect cell system under monolayer and suspension culture conditions. They concluded that the production of rHA1 using an insect suspension cell system may lead to the economical production of H5 antigen.
Read Full Text Article: Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture