Cat. # | Size | Qty. | Price |
---|---|---|---|
65895S | 100 µl (50 tests) |
|
REACTIVITY | H M |
SENSITIVITY | Endogenous |
MW (kDa) | |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Immunofluorescence (Immunocytochemistry) | 1:50 - 1:200 |
Flow Cytometry (Fixed/Permeabilized) | 1:400 |
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 182
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
Human, Mouse
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human PAX5 protein.
Paired box (PAX) proteins are a family of transcription factors that play important and diverse roles in animal development (1). Nine PAX proteins (PAX1-9) have been described in humans and other mammals. They are defined by the presence of an amino-terminal "paired" domain, consisting of two helix-turn-helix motifs, with DNA binding activity (2). PAX proteins are classified into four structurally distinct subgroups (I-IV) based on the absence or presence of a carboxy-terminal homeodomain and a central octapeptide region. Subgroup I (PAX1 and 9) contains the octapeptide but lacks the homeodomain; subgroup II (PAX2, 5, and 8) contains the octapeptide and a truncated homeodomain; subgroup III (PAX3 and 7) contains the octapeptide and a complete homeodomain; and subgroup IV (PAX4 and 6) contains a complete homeodomain but lacks the octapeptide region (2). PAX proteins play critically important roles in development by regulating transcriptional networks responsible for embryonic patterning and organogenesis (3); a subset of PAX proteins also maintain functional importance during postnatal development (4). Research studies have implicated genetic mutations that result in aberrant expression of PAX genes in a number of cancer subtypes (1-3), with members of subgroups II and III identified as potential mediators of tumor progression (2).
PAX5, also known as B cell-specific activator protein (BSAP), was originally identified as a DNA-binding protein with affinity for both immunoglobulin heavy-chain and kappa light-chain loci (5). PAX5 is unique within the PAX family in being the only member with reported expression in the hematopoietic system. PAX5 is required to promote differentiation of common lymphoid progenitors (CLPs) into B cells (5,6); it is also required for the continued maintenance of B cell identity following differentiation (7). Disruptions to the expression of PAX5 have consequently been linked with lymphoid cancer development (8).
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