NOS Clone 2A12-A4 Monoclonal Antibody
Anti-human inducible Nitric Oxide Synthase
Monoclonal Antibody 2A12-A4
Reactivity
HumanCrossreactivity Note: Cross reacts with nNOS and eNOS Maps to hiNOS (985-1002)
Applications
Immunoblot, Western Blot, Western Blot
Synonyms
NOS Clone 2A12-A4; Monoclonal Antibody; Anti-human inducible Nitric Oxide SynthaseMonoclonal Antibody 2A12-A4; anti-NOS Clone 2A12-A4 antibody
Reactivity
Human
Crossreactivity Note: Cross reacts with nNOS and eNOS Maps to hiNOS (985-1002)
Crossreactivity Note: Cross reacts with nNOS and eNOS Maps to hiNOS (985-1002)
Clonality
Monoclonal
Isotype
Mouse IgG1 kappa
Clone Number
2A12-A4
Specificity
Polypeptide ::: % Cross Reactivity
hiNOS (985-1002) ::: 100
rhiNOS (Type II) ::: 100
hiNOS (985-1002) ::: 100
rhiNOS (Type II) ::: 100
Form/Format
Supplied as Culture Supernatant
Applicable Applications for anti-NOS Clone 2A12-A4 antibody
Western Immunoblot, Western Blot, Immunofluorescent Staining of Induced Cells
Application Notes
Western Blot: Western immunoblots resulted in a single band being detected at ~ 130 kDa at a dilution of 1:20.
Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:20 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using three different cell lines. DLD-1(a human epithelial cell line). A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16-24 hours with a cytokine mixture or with a cytokine/LPS mixture. Following the treatment. the cells were washed x 3 and fixed in either neutral buffered formalin or 100% ice cold acetone. They were reacted for 60 minutes with the culture supernatant. and then with FITC-conjugated goat anti-mouse IgG. The immuno-fluorescent staining pattern was observed using epifluorescent microscopy.
Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:20 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using three different cell lines. DLD-1(a human epithelial cell line). A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16-24 hours with a cytokine mixture or with a cytokine/LPS mixture. Following the treatment. the cells were washed x 3 and fixed in either neutral buffered formalin or 100% ice cold acetone. They were reacted for 60 minutes with the culture supernatant. and then with FITC-conjugated goat anti-mouse IgG. The immuno-fluorescent staining pattern was observed using epifluorescent microscopy.
Western Blot Protocol: 1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels. such at 7.5% gels) and electrophoretic transfer to PVDF membrane. block the membrane overnight with 2% normal goat serum in TBS/Tween-20 buffer.
2. Wash x 2 with TBS/Tween-20.
3. Apply the mouse monoclonal antibody after preparing a 1:20 dilution. Use 2% normal goat serum in TBS/Tween-20 as buffer. and let the primary antibody bind for 2-4 hours.
4. Wash x 3 with TBS/Tween-20.
5. Apply affinity purified HRP-goat anti-rabbit IgG antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in TBS/Tween-20. Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques.
6. Wash x 4 for 5 min per wash in TBS/Tween-20 buffer.
7. Develop color using the enhanced DAB reaction.
Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:20 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using three different cell lines. DLD-1(a human epithelial cell line). A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16-24 hours with a cytokine mixture or with a cytokine/LPS mixture. Following the treatment. the cells were washed x 3 and fixed in either neutral buffered formalin or 100% ice cold acetone. They were reacted for 60 minutes with the culture supernatant. and then with FITC-conjugated goat anti-mouse IgG. The immuno-fluorescent staining pattern was observed using epifluorescent microscopy.
Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:20 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using three different cell lines. DLD-1(a human epithelial cell line). A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16-24 hours with a cytokine mixture or with a cytokine/LPS mixture. Following the treatment. the cells were washed x 3 and fixed in either neutral buffered formalin or 100% ice cold acetone. They were reacted for 60 minutes with the culture supernatant. and then with FITC-conjugated goat anti-mouse IgG. The immuno-fluorescent staining pattern was observed using epifluorescent microscopy.
Western Blot Protocol: 1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels. such at 7.5% gels) and electrophoretic transfer to PVDF membrane. block the membrane overnight with 2% normal goat serum in TBS/Tween-20 buffer.
2. Wash x 2 with TBS/Tween-20.
3. Apply the mouse monoclonal antibody after preparing a 1:20 dilution. Use 2% normal goat serum in TBS/Tween-20 as buffer. and let the primary antibody bind for 2-4 hours.
4. Wash x 3 with TBS/Tween-20.
5. Apply affinity purified HRP-goat anti-rabbit IgG antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in TBS/Tween-20. Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques.
6. Wash x 4 for 5 min per wash in TBS/Tween-20 buffer.
7. Develop color using the enhanced DAB reaction.
Related Product Information for anti-NOS Clone 2A12-A4 antibody
This culture supernatant contains mouse monoclonal antibody clone 2A12-A4 raised against recombinant hiNOS. It has been epitope mapped using 96 overlapping 18 amino acid long synthetic peptides which cover the entire 1153 amino acid length of hiNOS and was found to bind to a defined region of the hiNOS sequence. residues 985-1002. This monoclonal antibody has been found to stain hiNOS and ecNOS in western immunoblots and by immunocytochemistry. This monoclonal antibody was tested for recognition of other NOS isoforms by ELISA. western immunoblotting. and immunocytochemical techniques. It has been found to be a mouse IgG1 kappa by isotyping
Product Categories/Family for anti-NOS Clone 2A12-A4 antibody
NCBI and Uniprot Product Information
NCBI GI #
NCBI Official Full Name
NOS
Similar Products
Product Notes
The NOS Clone 2A12-A4 (Catalog #AAA350045) is an Antibody and is intended for research purposes only. The product is available for immediate purchase. The Anti-human inducible Nitric Oxide Synthase Monoclonal Antibody 2A12-A4 reacts with Human Crossreactivity Note: Cross reacts with nNOS and eNOS Maps to hiNOS (985-1002) and may cross-react with other species as described in the data sheet. AAA Biotech's NOS Clone 2A12-A4 can be used in a range of immunoassay formats including, but not limited to, Western Immunoblot, Western Blot, Immunofluorescent Staining of Induced Cells. Western Blot: Western immunoblots resulted in a single band being detected at ~ 130 kDa at a dilution of 1:20. Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:20 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using three different cell lines. DLD-1(a human epithelial cell line). A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16-24 hours with a cytokine mixture or with a cytokine/LPS mixture. Following the treatment. the cells were washed x 3 and fixed in either neutral buffered formalin or 100% ice cold acetone. They were reacted for 60 minutes with the culture supernatant. and then with FITC-conjugated goat anti-mouse IgG. The immuno-fluorescent staining pattern was observed using epifluorescent microscopy. Immunofluorescent: This monoclonal antibody has been found to stain cells induced to produce iNOS at a 1:20 dilution. The ability of this monoclonal antibody to bind to iNOS in fixed cells was examined using three different cell lines. DLD-1(a human epithelial cell line). A-172 (a human glioblastoma cell line) and RAW 264.7 (a mouse macrophage cell line). The cells were cultured for 2 days in normal medium and then induced to produce iNOS by treatment for 16-24 hours with a cytokine mixture or with a cytokine/LPS mixture. Following the treatment. the cells were washed x 3 and fixed in either neutral buffered formalin or 100% ice cold acetone. They were reacted for 60 minutes with the culture supernatant. and then with FITC-conjugated goat anti-mouse IgG. The immuno-fluorescent staining pattern was observed using epifluorescent microscopy. Western Blot Protocol: 1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels. such at 7.5% gels) and electrophoretic transfer to PVDF membrane. block the membrane overnight with 2% normal goat serum in TBS/Tween-20 buffer. 2. Wash x 2 with TBS/Tween-20. 3. Apply the mouse monoclonal antibody after preparing a 1:20 dilution. Use 2% normal goat serum in TBS/Tween-20 as buffer. and let the primary antibody bind for 2-4 hours. 4. Wash x 3 with TBS/Tween-20. 5. Apply affinity purified HRP-goat anti-rabbit IgG antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in TBS/Tween-20. Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques. 6. Wash x 4 for 5 min per wash in TBS/Tween-20 buffer. 7. Develop color using the enhanced DAB reaction. Researchers should empirically determine the suitability of the NOS Clone 2A12-A4 for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "NOS Clone 2A12-A4, Monoclonal Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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