ORDERING INFORMATION
Catalog Number: AF3080
Lot Number: WJV01
Size: 100 μg
Formulation: 0.2 μm filtered solution in PBS with 5% trehalose
Storage: -20° C
Reconstitution: sterile PBS
Specificity: human A33 extracellular domain
Immunogen: NS0-derived rhA33 extracellular domain
Ig Type: goat IgG
Applications: Western blot Immunohistochemistry
Flow Cytometry
Direct ELISA
HT-29 cells were stained with anti-A33 (R&D Systems, Cat. # AF3080, filled histogram) or control antibody (R&D Systems, Cat. # AB-108-C, open histogram) followed by PE-conjugated anti-goat antibody (R&D Systems, Cat. # F0107).
Preparation
Produced in goats immunized with purified, NS0-derived, recombinant human A33 (rhA33;    aa 22 - 235; Accession # Q99795). Human A33 specific IgG was purified by human A33 affinity chromatography. Human A33, also known as GPA33, is a 43 kDa type I transmembrane glycoprotein that belongs to the CTX (cortical thymocyte marker in Xenopus) family of cell adhesion molecules within the immunoglobulin superfamily. Other family members include CXADR, ESAM, BT-IgSF, CD2 and JAM A-C. The extracellular domain (ECD) of human A33 is 214 amino acids (aa) in length and contains one V-type and one C2-type Ig-like domain. This ECD is 80%, 74% and 71% aa identical to canine, bovine and mouse A33 ECD, respectively. A33 is likely to be involved in cell-cell adhesion between epithelial cells.
Formulation
Lyophilized from a 0.2 μm filtered solution in phosphate-buffered saline (PBS) with 5% trehalose.
Reconstitution
Reconstitute with sterile PBS. If 0.5 mL of PBS is used, the antibody concentration will be 0.2 mg/mL.
Storage
Lyophilized samples are stable for twelve months from date of receipt when stored at -20° C to -70° C. Upon reconstitution, the antibody can be stored at 2° - 8° C for 1 month without detectable loss of activity. Reconstituted antibody can also be aliquotted and stored frozen at -20° C to -70° C in a manual defrost freezer for six months without detectable loss of activity. Avoid repeated freeze-thaw cycles.
A33Cell Count
Specificity
This antibody has been selected for its ability to recognize human A33 in the applications listed below. In direct ELISAs and Western blots, this antibody shows approximately 10% cross-reactivity with rmA33.
Applications
Western blot - This antibody can be used at 0.1 - 0.2 μg/mL with the appropriate secondary reagents to detect human A33. The detection limit for rhA33 is approximately 2 ng/lane under non-reducing and reducing conditions.
Immunohistochemistry - This antibody will detect A33 in cells and tissues. The working dilution is 2 - 15 μg/mL. For chromogenic detection of labeling, use R&D Systems’ Cell and Tissue Staining Kits (CTS Series).
Flow Cytometry - This antibody was tested in flow cytometry using HT-29 cells. Dilute this antibody to 50 μg/mL and add 10 μL of this diluted solution to 1-2.5 x 105 cells in a total reaction volume not exceeding 200 μL. The binding of unlabeled antibodies may be visualized by adding a secondary developing reagent such as anti-goat IgG conjugated to a fluorochrome.
Direct ELISA - This antibody can be used at 0.5 - 1.0 μg/mL with the appropriate secondary reagents to detect human A33. The detection limit for rhA33 is approximately 0.2 ng/well.
Optimal dilutions should be determined by each laboratory for each application.

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