Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 65)

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2-D Electrophoresis Guide Appendices
Appendix A
Glossary
%C Cross-linker concentration; weight percentage of cross-linker in a polyacrylamide
gel. Effective pore size of a gel is a biphasic function of %C
%T Monomer concentration (acrylamide + cross-linker) in a gel (in g/100 ml).
Effective pore size of a gel is an inverse function of %T, and gels can be made
with a single, continuous %T throughout the gel (single-percentage gels), or they
can be cast with a gradient of %T through the gel (gradient gels)
2-D electrophoresis Two-dimensional electrophoresis. Proteins are separated first according to
isoelectric point (pI) by isoelectric focusing (IEF) and then according to size by
SDS-PAGE, yielding a two-dimensional protein map of spots
2-Mercaptoethanol Reducing agent used for cleavage of intra- and intermolecular disulfide bonds to
achieve complete protein unfolding and to maintain all proteins in a fully reduced
state. Also known as
b-mercaptoethanol or BME
Acrylamide Monomer used with a cross-linker to form the matrix used for separating proteins
or small DNA molecules
Ammonium persulfate Initiator used with TEMED (catalyst) to initiate the polymerization of acrylamide and
(APS) bisacrylamide in making a polyacrylamide gel; (NH
4
)
2
S
2
O
8
Ampholyte Amphoteric molecule that exists mostly as a zwitterion in a certain pH range.
Ampholytes are used to establish a stable pH gradient for use in isoelectric focusing
Amphoteric Containing both acidic and basic groups
Anode Positively charged electrode. Negatively charged molecules (anions) move towards
the anode, which is usually indicated by the color red
Anionic dye Negatively charged compound used as a stain; used in blotting to stain proteins
immobilized on membranes such as nitrocellulose or PVDF
Antibody Immunoglobulin (Ig); protein produced in response to an antigen, which specifically
binds the portion of the antigen that initiated its production
Assay Analysis of the quantity or characteristics of a substance
Background Nonspecific signal or noise that can interfere with the interpretation of valid signals
Bio-Spin ® columns Family of Bio-Rad sample preparation products that includes the Bio-Spin ® 6 and
Micro Bio-Spin ™ 6 columns; used for buffer exchange and desalting applications
Bis or bis-acrylamide A common cross-linker used with acrylamide to form a support matrix;
N,N'-methylene-bis-acrylamide
Blot Immobilization of proteins or other molecules onto a membrane, or a membrane
that has the molecules adsorbed onto its surface
Bromophenol blue Common tracking dye used to monitor the progress of electrophoresis
Carrier ampholytes Heterogeneous mixture of small (300–1,000 Da) polyamino-polycarboxylate
buffering compounds that have closely spaced pI values and high conductivity.
Within an electric field, they align according to pI to establish the pH gradient
Cathode Negatively charged electrode. Positively charged molecules (cations) move toward
the cathode, which is usually indicated by the color black
Chaotropic agent Chemical that disrupts inter- and intramolecular interactions (for example, urea
and thiourea)
CHAPS Zwitterionic detergent (having both positively and negatively charged groups
with a net charge of zero) that is widely used for protein solubilization for IEF and
2-D electrophoresis; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
Comb Object used to cast wells in an agarose or acrylamide gel. In PAGE applications,
square-bottom combs are inserted into the gel sandwich before polymerization
to form square-bottomed wells
Coomassie (Brilliant) Anionic dye used in the total protein staining of gels and blots and that comes in
Blue two forms: Coomassie (Brilliant) Blue G-250 differs from Coomassie (Brilliant) Blue
R-250 by the addition of two methyl groups
Criterion ™ cells, Family of Bio-Rad products used for midi-format vertical electrophoresis;
blotters, and gels includes the Criterion and Criterion ™ Dodeca ™ cells, Criterion blotter, and
Criterion precast gels
Cross-linker Molecule (for example, bis-acrylamide) used to link polymerizing monomer
molecules together to form a netlike structure within the gel. The holes in the nets
are called the pores, and the pore size is determined in part by the cross-linker
concentration. The pores may or may not sieve the macromolecules
Cup loading Application of protein sample onto IPG strips through sample cups applied to the
strips; can improve resolution at extremes of a pH gradient and improve uptake of
basic proteins
DC ™ assay kit Bio-Rad’s detergent-compatible protein assay kit
Depletion Reduction in the amount of high-abundance proteins relative to
low-abundance proteins
Discontinuous Electrophoresis gel system that uses different buffers and sometimes
buffer system different buffer compositions to focus and separate components of a sample.
Discontinuous systems typically focus the proteins into tighter bands than
continuous gel systems, allowing larger protein loads
Disulfide bond Chemical bond joining two sulfur atoms; commonly found in proteins,
contributing to their secondary and tertiary structures
Dithiotheithol (DTT) Reducing agent used for cleavage of intra- and intermolecular disulfide
bonds to achieve complete protein unfolding and to maintain all proteins in a fully
reduced state
Electrophoresis Movement of charged molecules in a uniform electric field
Equilibration Preparation of protein separated in an IPG strip for second-dimension SDS-PAGE;
reduces and alkylates sulfhydryl groups and saturates proteins with SDS
EXQuest ™ spot cutter Bio-Rad’s brand of spot cutter
Fractionation Separation of a sample into discrete parts for separate analysis; may improve
detection of low-abundance proteins and reduce sample complexity
Glycine Amino acid used as the trailing or slow ion in SDS-PAGE according to Laemmli
(Laemmli, 1970)
Gradient gel Gel with gradually changing monomer concentration (%T) in the direction of
migration. In SDS-PAGE, gradients are used to separate wider molecular weight
ranges of molecules than can be separated with single-percentage gels
Immobilized pH Strips in which buffering groups are covalently bound to an acrylamide gel
gradient (IPG) strips matrix, resulting in stable pH gradients. This eliminates problems of gradient
instability and poor sample loading capacity associated with carrier
ampholyte–generated pH gradients