Anti-p-Ubiquitin (pSer65) Antibody, clone 2N21 ZooMAb® Rabbit Monoclonal
SIGMA/ZRB1494 - recombinant, expressed in HEK 293 cells
Synonym: Ub
Product Type: Chemical
Immunocytochemistry Enhanced Validation-Recombinant Antibody Technology Immunofluorescent analysis of HeLa cells transfected with PINK was performed using a 1:100 dilution of Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the mitochondrial with treatment.
Immunocytochemistry Enhanced Validation-Recombinant Antibody Technology Immunofluorescent analysis of HeLa cells transfected with PINK was performed using a 1:100 dilution of Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the mitochondrial with treatment.
Immunocytochemistry Enhanced Validation-Recombinant Antibody Technology Immunofluorescent analysis of HeLa cells transfected with PINK was performed using a 1:100 dilution of Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the mitochondrial with treatment.
Immunocytochemistry Enhanced Validation-Recombinant Antibody Technology Immunofluorescent analysis of HeLa cells transfected with PINK and treated with Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was performed using a 1:100 dilution of Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the mitochondrial with treatment.
Immunocytochemistry Enhanced Validation-Recombinant Antibody Technology Immunofluorescent analysis of HeLa cells transfected with PINK and treated with Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was performed using a 1:100 dilution of Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the mitochondrial with treatment.
Immunocytochemistry Enhanced Validation-Recombinant Antibody Technology Immunofluorescent analysis of HeLa cells transfected with PINK and treated with Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was performed using a 1:100 dilution of Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the mitochondrial with treatment.
Western Blotting Enhanced Validation-Recombinant Antibody Technology Lysates from HeLa cells transfected with PINK (Lane 1) and HeLa cells transfected with PINK and treated with Carbonyl cyanide m-chlorophenyl hydrazine (CCCP; Lane 2) were probed with Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal (1:1,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Western Blotting Enhanced Validation-Recombinant Antibody Technology Lysates from PC3 cells and PC3 cells treated with Carbonyl cyanide m-chlorophenyl hydrazine (CCCP; Lane 2) were probed with Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal (1:1,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Affinity Binding Assay Enhanced Validation-Recombinant Antibody Technology 100 μg/mL of phosphorylated serine 65 Ubiquitin peptide (Run 1) or non-phosphorylated Ubiquitin control peptide (Run 2) were analyzed by affinity binding assay using Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal. The binding of this antibody to phosphorylated serine 65 Ubiquitin peptide displayed a KD value of 2.5 x 10-^8 whereas its binding to non-phosphorylated Ubiquitin control peptide displayed a KD of 2.4 x 104.
Flow Cytometry Enhanced Validation-Recombinant Antibody Technology Staining of one million PC-3 cells treated with Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was performed using 0.1 μg of Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal (Yellow histogram) or the equivalent amount of a Rabbit isotype control (Grey histogram), followed by a PE-conjugated Donkey Anti-Rabbit secondary antibody.
Peptide Inhibition Assay Enhanced Validation-Recombinant Antibody Technology HeLa transfected with PINK1 and treated with CCCP ( (15 mM, 5 h) were probed with Cat. No. ZRB1494, Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb® Rabbit Monoclonal (1:1,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system. Multiple bands indicate the extent of polyubiquitination. Lane 1: no peptide Lane 2: phosphorylated Ser 65 ubiquitin peptide Lane 3: non-phosphorylated ubiquitin control peptide
12 Principles of Green Chemistry: Principle 1—Waste Prevention. This product has a smaller environmental footprint through reduction of waste in the manufacturing process.
12 Principles of Green Chemistry: Principle 4—Designing Safer Chemicals
Chemical products should be designed to affect their desired function while minimizing their toxicity.
Chemical products should be designed to affect their desired function while minimizing their toxicity.
12 Principles of Green Chemistry: Principle 6—Design for Energy Efficiency. This product was re-engineered to utilize less energy in the manufacturing process.
| antibody form | purified antibody |
| antibody product type | primary antibodies |
| biological source | rabbit (recombinant) |
| clone | 2N21, monoclonal |
| recombinant monoclonal | |
| conjugate | unconjugated |
| enhanced validation | recombinant expression Learn more about Antibody Enhanced Validation  |
| form | lyophilized |
| greener alternative category | Aligned |
| greener alternative product characteristics | Waste Prevention Designing Safer Chemicals Design for Energy Efficiency Learn more about the Principles of Green Chemistry . |
| isotype | IgG |
| packaging | antibody small pack of 25 μL |
| product line | ZooMAb® learn more |
| recombinant | expressed in HEK 293 cells |
| shipped in | ambient |
| species reactivity | human |
| species reactivity (predicted by homology) | mouse, rat |
| storage temp. | 2-8°C |
| technique(s) | affinity binding assay: suitable |
| flow cytometry: suitable | |
| immunocytochemistry: suitable | |
| inhibition assay: suitable (peptide) | |
| western blot: suitable | |
| UniProt accession no. | Q16620  |
| Application: | Anti-p-Ubiquitin (pSer65), clone 2N21 ZooMAb, Cat. No. ZRB1494, is a recombinant Rabbit monoclonal antibody to detect ubiquitin (pSer65) in Affinity Binding Assay, Flow Cytometry, Immunocytochemistry, Peptide Inhibition Assay, and Western Blotting. |
| Application: | Western Blotting Analysis: A 1:1,000 dilution from a representative lot detected p-Ubiquitin (pSer65) in lysate from HeLa cells transfected with PINK and treated with CCCP. Flow Cytometry Analysis: 0.1 μg from a representative lot detected p-Ubiquitin (pSer65) in one million PC-3 cells treated with CCCP. Peptide Inhibition Assay: Target band detection in lysate from HeLa cells transfected with PINK and treated with CCCP (15 mM, 5 h) was prevented by preblocking of a representative lot with the phosphorylated Ser 65 ubiquitin peptide, but not by corresponding non-phosphopeptide. Immunocytochemistry Analysis: A 1:100 dilution from a representative lot detected p-Ubiquitin (pSer65) in HeLa cells transfected with PINK and treated wtih CCCP. Affinity Binding Assay: A representative lot of this antibody bound phosphorylated serine 65 Ubiquitin peptide with a KD of 2.5 x 10-8 in an affinity binding assay. Note: Actual optimal working dilutions must be determined by end user as specimens, and experimental conditions may vary with the end user |
| Disclaimer: | Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. |
| General description: | We are committed to bringing you greener alternative products, which adhere to one or more of The 12 Principles of Green Chemistry.This antibody is Preservative-free, produced without the harm or sacrifice of animals and exceptionally stable to allow for ambient shipping and storage if needed and thus aligns with "Waste Prevention", "Designing Safer Chemicals" and "Design for Energy Efficiency". Click here for more information. |
| General description: | ZooMAb antibodies represent an entirely new generation of recombinant monoclonal antibodies. Each ZooMAb antibody is manufactured using our proprietary recombinant expression system, purified to homogeneity, and precisely dispensed to produce robust and highly reproducible lot-to-lot consistency. Only top-performing clones are released for use by researchers. Each antibody is validated for high specificity and affinity across multiple applications, including its most commonly used application. ZooMAb antibodies are reliably available and ready to ship when you need them. Learn more about ZooMAb here. |
| Immunogen: | KLH-conjugated linear peptide corresponding to 13 amino acids from the ubiquitin-like 1 domain of human polyubiquitin phosphorylated on serine 65. |
| Legal Information: | ZooMAb is a registered trademark of Merck KGaA, Darmstadt, Germany |
| Physical form: | Purified recombinant rabbit monoclonal antibody IgG, lyophilized in PBS with 5% Trehalose, normal appearance a coarse or translucent resin. Contains no biocide or preservatives, such as azide, or any animal by-products. Larger pack sizes provided as multiples of 25 μL. |
| Reconstitution: | 0.03 mg/mL after reconstitution at 25 μL per vial. Please refer to guidance on suggested starting dilutions and/or titers per application and sample type. |
| Specificity: | Clone 2N21 is a ZooMAb rabbit recombinant monoclonal antibody that specifically detects ubiquitin phosphorylated on serine 65. |
| Storage and Stability: | Recommend storage of lyophilized product at 2-8°C; Before reconstitution, micro-centrifuge vials briefly to spin down material to bottom of the vial; Reconstitute each vial by adding 25 μL of filtered lab grade water or PBS; Reconstituted antibodies can be stored at 2-8°C, or -20°C for long term storage. Avoid repeated freeze-thaws. |
| Target description: | BDNF/NT-3 growth factors receptor (UniProt: Q16620; also known as EC:2.7.10.1, GP145-TrkB, Trk-B, Neurotrophic tyrosine kinase receptor type 2, TrkB tyrosine kinase, Tropomyosin-related kinase B) is encoded by the NTRK2 (also known as TRKB) gene (Gene ID: 4915) in human. TrkB is a single-pass type I membrane protein that is internalized to endosomes upon ligand-binding. It is synthesized with a signal peptide (aa 1-31) that is subsequently cleaved off to generate the mature form that contains an extracellular domain (aa 32-430), a transmembrane domain (aa 431-454), and a cytoplasmic domain (aa 455-822). TrkB is a receptor tyrosine kinase that is involved in the development and maturation of the central and the peripheral nervous systems through regulation of neuron survival, proliferation, migration, differentiation, and synapse formation and plasticity. In the central nervous system, its expression is observed in the cerebral cortex, hippocampus, thalamus, choroid plexus, granular layer of the cerebellum, brain stem, and spinal cord. In the peripheral nervous system, it is expressed in many cranial ganglia, the ophthalmic nerve, the vestibula, and multiple facial structures. Upon ligand-binding, it undergoes homodimerization, autophosphorylation, and activation. It can then recruit, phosphorylate and/or activate several downstream effectors including SHC1, FRS2, SH2B1, SH2B2 and PLCG1 that regulate distinct overlapping signaling cascades. TrkB is also reported to play a role in neutrophin-dependent calcium signaling in glial cells and mediate communication between neurons and glia. Following binding of BDNF to TrkB receptor, Tyrosine 816 is phosphorylated, which allows the binding of phospholipase C gamma and activation of signaling cascade. This ZooMAb recombinant monoclonal antibody, generated by our propriety technology, offers significantly enhanced specificity, affinity, reproducibility, and stability over conventional monoclonals. |
| WGK Germany | WGK 1 |
| Flash Point(F) | Not applicable |
| Flash Point(C) | Not applicable |
| Storage Temp. | 2-8°C |
| UNSPSC | 12352203 |