The Mini Trans-Blot cell can transfer up to two mini gels (10 x 7.5 cm) in an hour and is available either as a complete apparatus or as a module that uses the buffer tank and lid of the Mini-PROTEAN ® Tetra cell for operation. The Mini Trans-Blot cell and the Criterion Blotter accommodate mini- and midi-format gels, respectively. Mini Trans-Blot ® Cell and Criterion™ Blotter These cooling systems prevent temperature fluctuations and overheating during high-intensity, extended, or native protein transfers
Cooling mechanism - cooling systems consist of an ice block, a sealed ice unit, or a cooling coil that is coupled to an external cooling mechanism. Plate electrodes offer greater field strength than wire electrodes but wire electrodes may be more economical and generate less heat Electrodes - tank transfer systems use either plate or wire electrode cards. Cassettes are made of non-conductive material and designed to permit unimpeded flow of current and buffer through the gel and membrane sandwich Gel holder cassette - the gel and membrane sandwich is held together between two foam pad and filter paper sheets, and placed into the tank within a gel holder cassette. Ports on the lid allow connection points for the electrodes and are energized using an external power supply On the inside, the tank has slots for placement of the electrode cards, gel holder cassettes, and cooling element. Buffer tank and lid - the buffer tank and lid combine to fully enclose the inner chamber during electrophoresis. Tank transfer systems contain the following elements: Selection of the appropriate system is largely dictated by the gel format used for separation and the desired throughput. More non-specific signal can arise from the binding of the secondary antibody to other proteins on the blot.Įxtra incubation and wash steps add time to the experiment.The tank transfer systems offered by Bio-Rad are described below, and their specifications are summarized in the table below. Labeling every primary antibody adds time and cost. Provides access to a wider range of labels.Ĭoupling of label to the primary antibody may affect the antibody’s ability to bind to the target protein. Saves labeling time and expense, especially when all primary antibodies are made in the same species. Often gives a stronger signal because multiple secondary antibodies bind to each primary antibody.Įasy to change label type or detection methods for a new experiment by swapping secondaries. Indirect Detection.Ĭomparison of Direct and Indirect Detection Methodsįaster overall, since there are fewer steps. Biotinylated primary antibodies also require a two-step detection procedure however, the second step involves incubation with streptavidin, a bacterial protein, conjugated to HrP (or AP), rather than with a labeled antibody.įigure 11: Direct vs. For example, one of our popular secondaries is STAr88P, a purified donkey antibody raised against goat/sheep immunoglobulin (IgG) which is coupled to an HrP label. The labeled secondary antibody is typically directed against the immunoglobulin class or subclass of the primary antibody’s species. After washing, a labeled secondary antibody is used to detect the presence of the primary antibody, and thus the target protein. First, the Western blot is incubated with an unlabeled primary antibody directed against the target protein. With indirect detection, two different antibodies are used in sequence for the detection step. In addition, it is possible to directly label an antibody by using a commercially supplied labeling kit such as our LYNX Rapid Conjugation Kits®, or with in-house reagents. 
Labeled primary antibodies can be ordered directly from Bio-Rad or other commercial antibody suppliers.
The one-step procedure, direct detection, relies upon a single antibody which has been covalently joined to an easily detected label molecule (biotin, an enzyme, or a fluorescent dye). Antibody detection of the target protein is accomplished using a one-step or two-step protocol.
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