Fc gamma RIIIA/CD16a Overexpression Lysate

Novus Biologicals | Catalog # NBP2-11370

Novus Biologicals
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Key Product Details

Species

Human

Applications

Western Blot

Product Summary for Fc gamma RIIIA/CD16a Overexpression Lysate

Fc gamma RIIIA/CD16a Transient Overexpression Lysate


Expression Host: HEK293T

Plasmid: RC225332

Accession#: NM_001127593

Protein Tag: C-MYC/DDK

You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.

Product Specifications

Application Notes

This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.

Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.

Theoretical MW

27.2 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Type

Overexpression

Scientific Data Images for Fc gamma RIIIA/CD16a Overexpression Lysate

Western Blot: Fc gamma RIIIA/CD16a Overexpression Lysate [NBP2-11370]

Western Blot: Fc gamma RIIIA/CD16a Overexpression Lysate [NBP2-11370]

Western Blot: CD16 Overexpression Lysate (Adult Normal) [NBP2-11370] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for CD16.

Formulation, Preparation, and Storage

Formulation

RIPA buffer

Concentration

The exact concentration of the protein of interest cannot be determined for overexpression lysates. Please contact technical support for more information.

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store at -80C. Avoid freeze-thaw cycles.

Background: Fc gamma RIIIA/CD16a

Fc gamma receptor IIIA (RIIIA), also called CD16a, is an activating natural killer (NK) cell receptor that binds the Fc portion of immunoglobulin G (IgG) antibodies and is responsible for eliciting a host defense against microbial pathogens (1). Fc gamma RIIIA/CD16a belongs to the Fc gamma RIII (CD16) subclass of Fc gamma receptors (1,2). The two other subclasses of receptors are Fc gamma RI (CD64) and Fc gamma RII (CD32) (1,2). The two form of Fc gamma RIII are Fc gamma RIIIA (CD16a) and Fc gamma RIIIB (CD16b), which are encoded by two different homologous genes, FCGR3A and FCGR3B, respectively (1-3). The human Fc gamma RIIIA protein is 254 amino acids (aa) in length with a theoretical molecular weight (MW) of 29 kDa (1,4,5). CD16a contains five glycosylation sites, two disulfide bonds, and two Ig-like C2 domains (4). Fc gamma RIIIA/CD16a is expressed as a transmembrane protein on NK cells and on a subset of monocytes, macrophages, CD4+ T cells, basophils, and mast cells (1-3). The soluble form of CD16 (sCD16) is often produced following exposure to inflammatory signals and protein shedding via metalloproteinases (3). Reduced sCD16 levels have been found in patients with multiple myeloma (3).

Activating NK cell receptor function has been harnessed for its potential in tumor immunotherapy (5). One immunotherapy strategy is using bi- and tri-specific NK cell engagers (BiKE and TriKE) to target the Fc gamma RIIIA/CD16a receptor with tumor-associated antigens to stimulate a cytotoxic response and mount an attack on tumor cells (5). CD16a is also capable of antibody dependent cellular cytotoxicity (ADCC) through recognition of antibodies bound to target cells (5-6). CD16-induced NK cell activation allows for NK co-receptor expression including stimulatory receptors like CD137 or inhibitory receptors like TIGIT and PD-1, which serve as additional regulatory checkpoints during ADCC (6). Therapeutic antibodies for cancer treatment like rituximab or trastuzumab can be recognized by Fc gamma RIIIA/CD16a to activate NK cell-mediated killing of tumor cells (5-6).

References

1. Fossati, G., Bucknall, R. C., & Edwards, S. W. (2001). Fcgamma receptors in autoimmune diseases. European Journal of Clinical Investigation, 31(9), 821-831. https://doi.org/10.1046/j.1365-2362.2001.00881.x

2. Patel, K. R., Roberts, J. T., & Barb, A. W. (2019). Multiple Variables at the Leukocyte Cell Surface Impact Fc gamma Receptor-Dependent Mechanisms. Frontiers in Immunology, 10, 223. https://doi.org/10.3389/fimmu.2019.00223

3. Moldovan, I., Galon, J., Maridonneau-Parini, I., Roman Roman, S., Mathiot, C., Fridman, W. H., & Sautes-Fridman, C. (1999). Regulation of production of soluble Fc gamma receptors type III in normal and pathological conditions. Immunology Letters, 68(1), 125-134. https://doi.org/10.1016/s0165-2478(99)00041-3

4. Uniprot (P08637)

5. Sivori, S., Pende, D., Quatrini, L., Pietra, G., Della Chiesa, M., Vacca, P., Tumino, N., Moretta, F., Mingari, M. C., Locatelli, F., & Moretta, L. (2021). NK cells and ILCs in tumor immunotherapy. Molecular Aspects of Medicine, 80, 100870. https://doi.org/10.1016/j.mam.2020.100870

6. Muntasell, A., Ochoa, M. C., Cordeiro, L., Berraondo, P., Lopez-Diaz de Cerio, A., Cabo, M., Lopez-Botet, M., & Melero, I. (2017). Targeting NK-cell checkpoints for cancer immunotherapy. Current Opinion in Immunology, 45, 73-81. https://doi.org/10.1016/j.coi.2017.01.003

Long Name

Fc gamma Receptor III A

Alternate Names

CD16a, FCGR3A, FcgRIIIA

Gene Symbol

FCGR3A

Additional Fc gamma RIIIA/CD16a Products

Product Documents for Fc gamma RIIIA/CD16a Overexpression Lysate

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Product Specific Notices for Fc gamma RIIIA/CD16a Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

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FAQs for Fc gamma RIIIA/CD16a Overexpression Lysate

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  • Q: I am interested in developing a surrogate ADCC assay (FC gamma III a binding assay) for which, I would require a CD16 Recombinant Protein as an antigen and Anti-Human IgG (Fc specific) as a secondary antibody as I am planning to perform this assay in an ELISA format. With this brief description, could you please suggest me the right CD16 Recombinant Protein among the three listed products in your web site (www.novusbio.com/proteins/cd16). Appreciate if you can share a reference article, where the use of this protein is cited.

    A: The right CD16 protein for you depends on what sort of functionality you require for your assay. H00002214-P01 is made by a Taiwanese company called Abnova and we distribute it for them. They do not test the activity of their recombinant proteins. The proteins are produced in a cell-free wheat germ system with an N-terminal 26kDa GST-tag. Due to this expression system, in our experience, the proteins do not always undergo the same folding and post-transnational modifications they might undergo in an endogenous cell. Because of this, we do not recommend this protein for use in functional assays and do not guarantee them for this use. They are mainly intended for use in Western Blots and ELISA as a positive control with their antibody. NBP1-98908 is produced in E. coli and contains a His-tag and it also not guaranteed for functionality but may be due to its E. coli expression system. H00002214-G01 is also an Abnova and is made in their cell-free wheat germ expression system with proprietary liposome technology. This is a recently developed system by them and is intended to help the proteins fold and maintain functionality. Which protein would be best for you depends on what you require for your assay. Here is a link to our antibodies to the Fc region of human IgG. The best antibody for you will depend on what you would like to use for detection.

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