Home Technique / Proteomics and protein biochemistry / Protein electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (introduction) new protocol
This is an introduction of 2D-gel from Wellcome Trust. ...

Buffer Recipes For 2D electrophoresis (pdf) new protocol
A very detailed protocol on preparing 2D electrophoresis and many useful tips. Protocol is in pdf format ...

Protein electrophoresis buffer recipes and running condition protocol
Some buffer recipes and running conditions for protein electrophoresis from invitrogen.(link changed, last modified on 6/11/05) (Invitrogen) ...

Buffer recipes for making SDS polyacrylamide gel protocol
This is a buffer recipe for making SDS-Polyacrylamide (SDS-PAGE) gels. It uses 40% polyacrylamide solution to cast 6%, 7.5%, 8%, 10% and 12% SDS-PAGE gels for SDS-PAGE electrophoresis. ( Breeden lab, Fred Hutchinson Cancer Research Center) ...

Protein Extraction from Polyacrylamide Gel using GeBAflex-tube protocol
GeBAflex-tube gel extraction kit is based on a single-use tube style, single step for rapid extraction of Protein, RNA, DNA, and complexes of RNA-proteins, DNA-protein and Protein-Protein from any gel. (GeBA) ...

Purify proteins from polyacrylamide gels protocol
Pierce protocol on protein purification from polyacrylamide gels. (Pierece) ...

Polyacrylamide Gel Drying Procedure protocol
A brief polyacrylamide gel drying procedure from Finkbeiner lab, UCSF. ...

Analysis of Two-Dimensional Electrophoresis Gel Images protocol
A thesis (pdf) by Lars Pedersen describes and proposes solutions to some of the currently most important problems in pattern recognition and image analysis of two-dimensional gel electrophoresis (2DGE) images. (Lars Pedersen, Technical University of Denmark) ...

Coomassie Brilliant Blue staining of proteins on PVDF new protocol
An PVDF membrane protein staining protocol from NCBA, China. ...

2D electrophoresis for proteomics tutorial new recommended review
Tutorial written by Dr James R. Jefferies, Parasitology Group, Institute of Biological Sciences, University of Wales at Aberystwyth, UK. ...

2D gele electrophoresis for proteomics tutorial new recommended review
A tutorial by Dr James R. Jefferies ( Parasitology Group, Institute of Biological Sciences, University of Wales at Aberystwyth). Content: 1. What is 2D Gel Electrophoresis? 2. What is it used for? 3. How is it Performed? 4. How is t ...

Native non-denaturing gel electrophoresis new hot review
Nice introduction on native or non-denaturing gel electrophoresis with gel images. (Alliance Protein Laboratories Inc.) "Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDS-PAGE the electrophoretic mobility of proteins ...

Protein electrophoresis new review
Protein electrophoresis overview 1. SDS-PAGE electrophoresis: use to determine the molecular weight of proteins. Measuring Molecular Weight with SDS-PAGE (LogMW versus the relat ...

SDS-PAGE gel electrophoresis problems new recommended troubleshooting
The "Hall of Shame" presents examples of some of the worst gels students have run in past labs. They represent many of the ways one can mess up a gel (but not all of them - we're still finding new ways!). See what features of your own gel(s) were unsatisfactor ...

Resolving marginally different proteins on SDS-PAGE gel electrophoresis top rated troubleshooting
We occasionally need to resolve hyper- and under-phosphorylated forms of a protein (pRb) on SDS-PAGE gels for Western blot analysis. Individuals familiar with these isoforms will know that they differ by an apparent (maximum of) 6 Kd, and separation i ...

Heavy and light chains on Coomaasie gel troubleshooting
Coomassie Blue Staining of SDS-PAGE Gel Protocol for coomassie blue staining of polyacrylamide gels: Coomassie Blue Staining of SDS-PAGE Gel ...

Making own gel dryer troubleshooting
How can I make my own gel dryer for plyacrylamide gels? Already have vacuum pump and oven. ... ... ...

Carbonate-based blotting transfer solution recipie? troubleshooting
A while ago I've seen a post that referenced a paper about the subject. The claim was it is a lot cheaper and works better than Tris/Glycine and on a par with CAPS-based solutions. We do awful lot of Westerns and although the most expensive part of ...

protein electrophoresis problem troubleshooting
I have a simple question, but it's hard for me. I run SDS-PAGE using the protein samples prepared by myself, and stain the gel with commassie blue after ecletrophoresis. The troublesome thing is that there is always smear on the gel according to the p ...

Ammonium sulfate and SDS (and more) troubleshooting
I have some protein samples that were electroeluted from a SDS-polyacrylamide gel, so they are with SDS and the usual components of the gel and the running buffer. I want to remove all these components as I want to perform a digestion of thes ...

SDS-PAGE running buffer exhaustion troubleshooting
Does anyone know how to calculate for how long an SDS-PAGE electrophoresis buffer can be re-used. I remember a talk at a conference where the speaker calculated for how long a electrophoresis buffer could be used before exhaustion. Any ... ... ...

Guidlines of sample preparation for 2-D electrophoresis new troubleshooting
University of Pennsylvania, Genomics institute, Proteomics core facility guidline on 2DE sample preparation. ...

Troubleshooting 2d gels (2D electrophoresis) new troubleshooting
This troubleshooting guide details common problems observed when performing gel electrophoresis and suggests ways in which they can be avoided. (Dr. Tony Dixon, the University of Leeds) Content: 1. No distinct spots are visible 2. Individual pro ...

Protein electrophoresis tips and troubleshooting new troubleshooting
Some tips (buffers and gel characteristics etc) and troubleshooting guide on protein gel electrophoresis by Les Lane. ...

SUMOplot: prediction of attachment of SUMO protein (11kDa) at multiple positions new database
SUMOplot™ can help you to explain larger MWs than expected on SDS gels due to attachment of SUMO protein (11kDa) at multiple positions of your protein. (Abgent Inc.) SUMO-1 (small ubiquitin-related modifier; also known as PIC1, UBL1, Sentrin, GMP1, an ...

2D Gel Electrophoresis Interactive Tutorial new recommended media
The rising interest in proteomics has catapulted 2D gel electrophoresis to the forefront of modern research techniques. It can be used to analyze global protein expression patterns or used to examine the differential expression of proteins in comparable sample ...

More electrophoresis protocols

SDS-PAGE (PolyAcrylamide Gel Electrophoresis)
(Davidson college)
Molecular biology

One-dimensional SDS gel electrophoresis of proteins.
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.1.)
One-dimensional SDS gel electrophoresis of proteins.

Gallagher SR.

Hoefer Pharmacia Biotech, San Francisco, California, USA.

Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit contains protocols that gives the standard Laemmli method for discontinuous gel electrophoresis under denaturing conditions, and the standard method for full-size gels is adapted for the minigel format. Minigels provide rapid separation but give lower resolution. Several alternate protocols are provided for specific applications, including electrophoresis of peptides and small proteins, continuous SDS-PAGE, ultrathin gels, multiple single-concentration gels, gradient gels, multiple gradient gels, and multiple gradient minigels.

Protein Ladder
Storage of protein marker ladder

Electrophoresis technique books
(Biowww bookshelf)
Electrophoresis technique related books

protein purification from acrylamide gel
need protocol to purify a protein band from a SDS-PAGE and then elute it

Laemmli buffer composition for diluting protein
composition of Laemmli buffer for protein electrophoresis.

no bands in SDS-PAGE
Protein electrophoresis problem

Capillary electrophoresis of peptides and proteins using isoelectric buffers.
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.13.)
Righetti PG, Bossi A, Gelfi C.

University of Verona, Verona, Italy.

Capillary electrophoresis in acidic isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and no interaction of macromolecules with the untreated wall ensues. In addition, because of the low conductivity of quasi-stationary isoelectric buffers, high voltage gradients can be applied (up to 800 V/cm), permitting fast peptide analysis with a high resolving power and minimal diffusional peak spreading.

Capillary electrophoresis of proteins and peptides
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.9.)
Burgi D, Smith AJ.

Genomyx, Foster City, California, USA.

Capillary electrophoresis (CE) is a high-resolution technique for the separation of a wide variety of molecules of biological interest such as metabolites, drugs, amino acids, nucleic acids, and carbohydrates. This unit focuses on the use of CE to separate proteins and peptides based on charge-to-mass ratios. Separation of proteins based on their isoelectric points is described along with a protocol for optimizing the separation conditions for a given protein. CE is also used for separations of peptides on an analytical scale and as a micropreparative technique--with either multiple separations that are pooled or a single, larger-scale separation--for the isolation of peptides from a protease digestion. In most of these examples the same capillary column can be used for all the separations. Only changes in buffer composition, ionic strength, and the presence or absence of additives are required for each specific application.

One-dimensional electrophoresis using nondenaturing conditions
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.3.)
Gallagher SR.

Hoefer Pharmacia Biotech, San Francisco, California, USA.

Nondenaturing or "native" electrophoresis--i.e., electrophoresis in the absence of denaturants such as detergents and urea--is an often-overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Two protocols are presented in this unit: continuous PAGE, which is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH, and discontinuous PAGE, which is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.

Transverse urea-gradient gel electrophoresis
(Curr Protoc Protein Sci. 2001 May;Chapter 7:Unit 7.4.)
Goldenberg DP.

University of Utah, Salt Lake City, Utah, USA.

Monitoring the cooperative unfolding transition induced when a protein is exposed to elevated temperature or a chemical denaturant is an important strategy for characterizing the conformational properties of a globular protein. This transition may be analyzed quantitatively by a variety of spectroscopic techniques, but a simpler alternative is described in this unit: urea-gradient gel electrophoresis. The pattern produced in the resulting gel can be used to estimate both the free energy change for unfolding and the rate of the unfolding transition. In addition, the technique can help identify either covalent or conformational heterogeneity in a protein sample. Because urea-gradient gel patterns are sensitive to several parameters, including hydrodynamic volume, net charge, and conformational stability, the technique can be particularly useful for comparing two forms of a protein, e.g., a natural form and the product of recombinant bacteria.

Protein profiling using two-dimensional difference gel electrophoresis (2-D DIGE)
(Curr Protoc Protein Sci. 2003 Feb;Chapter 22:Unit 22.2.)
Lilley KS.

University of Cambridge, Cambridge, United Kingdom.

2D-DIGE relies on pre-electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one gel. The dye-labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with spot matching between gels. Image analysis programs can then be used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable expression differences to be identified and quantified. This unit describes the DIGE procedure in terms of sample preparation from various types of cells, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples.

Two-dimensional gel electrophoresis
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.4.)
Harper S, Mozdzanowski J, Speicher D.

The Wistar Institute, Philadelphia, Pennsylvania, USA.

Two-dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two-dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS-PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two-dimensional gels, and two-dimensional protein databases.

Preparing protein extracts for quantitative two-dimensional gel comparison.
(Curr Protoc Protein Sci. 2004 Aug;Chapter 22:Unit 22.4.)
Chevallet M, Tastet C, Luche S, Rabilloud T.

Départment Résponse et Dynamique, Cellulaire/BioEnergetique Cellulaire et Pathologique, Commissariat à l'Energie Atomique, Grenoble, France.

This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample-dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for sample-preparation optimization. These solubilization procedures take into account the constraints brought by two-dimensional electrophoresis and are thus well suited for proteomic approaches.

Protein charge determination
(Curr Protoc Protein Sci. 2005 Sep;Chapter 2:Unit 2.10)
Winzor DJ.

University of Queensland, Brisbane, Queensland.

The most popular current method of determining protein valence entails the calculation of net charge from amino acid sequence/composition. However, the inaccuracy of that approach was recognized long before the advent of the protein data banks and computer programs to facilitate its adoption. Capillary zone electrophoresis affords the simplest and most economical procedure for obtaining a reliable estimate of the net charge of a protein in the buffer system of interest. This unit explains the major pitfalls in the calculation of net charge from protein sequence data.

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