High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Analytical Biochemistry
M W Bolt, P A Mahoney

Abstract

High-molecular-weight proteins often blot onto nitrocellulose membranes poorly following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting in low levels of detection on immunoblots, and hence difficulty in analyzing rare proteins. Moreover, optimizing conditions for the transfer of high-molecular-weight proteins to nitrocellulose frequently results in the inefficient transfer or loss of lower molecular weight proteins. This problem is particularly vexing during the analysis of large proteins which may be processed to one or more smaller biologically active forms. Using radiolabeled protein standards and phosphorimaging technology, we have quantitated the efficacy of many different protein gel electrophoresis and blotting protocols. Here we report novel gel and blotting conditions which significantly improve the transfer and retention of high-molecular-weight proteins, without sacrificing the efficient transfer of lower molecular weight proteins. Using this newly described procedure, we have detected a rare 500-kDa protein in immunoblots which was previously not detectable with any of the commonly used blotting procedures. Since the improved conditions offer increased sensitivity across a spectrum of protein s...Continue Reading

Citations

Aug 18, 2012·Genes & Development·Michalina JaniszewskaIvan Stamenkovic
Jun 29, 2010·Journal of Bacteriology·Anja NenningerConrad W Mullineaux
Nov 27, 2009·Canadian Journal of Microbiology·Fan ChenXing-Guo Wang
Apr 17, 2014·BioMed Research International·Sean C Taylor, Anton Posch
May 20, 2014·Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology·Patrícia AnacletoRui Rosa
Feb 13, 2016·Analytical Biochemistry·Jack O'Sullivan, Hilary E M McMahon
Dec 29, 2015·Behavioural Brain Research·Mohammad HaddadiS R Ramesh
Sep 14, 2010·Neuroscience Research·Shukuko YoshidaMotohiro Okada
Dec 17, 2009·Journal of Pharmacological and Toxicological Methods·Daniel J MacPhee
Jan 20, 2015·Proteomics. Clinical Applications·Thomas A Gorr, Johannes Vogel
Apr 5, 2013·Liver Transplantation : Official Publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society·Naina ShahRajiv Jalan
May 17, 2014·The Anatomical Record : Advances in Integrative Anatomy and Evolutionary Biology·J E RupertM T Butcher
Jun 14, 2005·Experimental Cell Research·Adrian J McNairnDavid M Gilbert
Aug 31, 2002·Analytical Biochemistry·Willy V BienvenutDenis F Hochstrasser
Jun 14, 2005·Analytical Biochemistry·Richard W Fitch, John W Daly
Jan 21, 2000·Mechanisms of Development·M GrammontJ L Couderc
Jun 19, 2013·Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology·D MadeiraM S Diniz
Feb 7, 2012·Behavioural Brain Research·Randi-Michelle CowinRichard Paylor
Jan 19, 2011·Analytical Biochemistry·Daniel PanAnna Tan-Wilson
Apr 15, 2014·World Journal of Urology·Masataka SunagawaLori A Birder
Jan 15, 2015·Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology·Diana MadeiraMário S Diniz
Jan 10, 2002·Genesis : the Journal of Genetics and Development·A DebecJ L Couderc
May 1, 2001·Current Protocols in Toxicology·M K Bruno, S D Cohen
Aug 28, 2007·Electrophoresis·Edita AksamitieneAnatoly Kiyatkin
Apr 17, 2008·Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology·Stefanie StiebSven Schenk
Sep 16, 2005·Journal of Cellular Biochemistry·Adrian J McNairn, David M Gilbert
Nov 26, 2015·Nature Communications·Daniel Nunes, Thomas Kuner

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.