A mouse monoclonal, anti-human PSMA antibody (Novus Biological, NBP1-45057, Littleton, CO) was used for western blot analysis and immunohistochemistry (IHC) and a mouse monoclonal, anti-human PSMA antibody (Abcam, ab19071, Cambridge, UK) was used for confocal fluorescent microscopy experiments. An anti-β actin antibody (Abcam) was used as a loading control for western blot analysis. ProLong Gold Antifade Mount (P10144), 4’, 6-diamidino-2-phenylindoleg (DAPI), and goat anti-mouse Alexa 488 secondary antibody (A-11034) were purchased from ThermoFisher Scientific (Waltham, MA). Diva (DV2004), Peroxidazed 1 (PX968), Background Punisher (BP974), secondary antibodies (MC541 and IPK5010), and hematoxylin (CATHE) used in IHC were purchased from commercial vendors.
Nbp1 45057
Manufactured by Novus Biologicals
NBP1-45057 is a recombinant protein product manufactured by Novus Biologicals. It is a purified protein that can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab19071, by Abcam (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Ab6994, by Abcam (1 mentions)
Ab53554, by Abcam (1 mentions)
Ab133357, by Abcam (1 mentions)
Ab58779, by Abcam (1 mentions)
Lmd6000, by Leica Microsystems (1 mentions)
Aida version 4, by Raytest (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Intellipath flx, by Biocare Medical (1 mentions)
Xrs molecular imager system, by Bio-Rad (1 mentions)
5 protocols using nbp1 45057
1
Evaluation of PSMA Antibodies for Western Blot, IHC, and Confocal Microscopy
2
Multimodal Assessment of Brain Pathology
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Cryosections of the brains were stained with DAPI (4′,6-diamidino-2-phenylindole) and evaluated by fluorescence microscopy. Disturbance of the BBB was visually evaluated using Evans blue dye (EBD). Therefore, 500 μl/kg 2% EBD was injected i.v. 30 min before decapitation of the rat for AR. EBD extravasation in brain slices was examined by fluorescence microscopy (LMD6000, Leica Microsystems CMS GmbH) and processed by AIDA software (AIDA Version 4.50, Raytest).
Blood vessels were stained with anti-rat von Willebrand factor antibody (ab6994, Abcam). To visualize activated microglia, anti-rat CD11b (Integrin alpha M) antibody (ab133357, Abcam) was used. Reactive astrocytes were visualized by staining with anti-rat GFAP (glial fibrillary acidic protein) antibody (ab53554, Abcam). Immunofluorescence PSMA staining was applied to tumor bearing brain slices using three different anti-PSMA antibodies (NBP1-45057 and NBP1-89822, Novus Biologicals; ab58779, Abcam) to display PSMA expression. To verify the functionality and specificity of the used PSMA antibodies, rat prostate and kidney tissue were used as positive controls. Immunofluorescence stainings were performed according to standard histology protocols and as described before [19 (link)].
Blood vessels were stained with anti-rat von Willebrand factor antibody (ab6994, Abcam). To visualize activated microglia, anti-rat CD11b (Integrin alpha M) antibody (ab133357, Abcam) was used. Reactive astrocytes were visualized by staining with anti-rat GFAP (glial fibrillary acidic protein) antibody (ab53554, Abcam). Immunofluorescence PSMA staining was applied to tumor bearing brain slices using three different anti-PSMA antibodies (NBP1-45057 and NBP1-89822, Novus Biologicals; ab58779, Abcam) to display PSMA expression. To verify the functionality and specificity of the used PSMA antibodies, rat prostate and kidney tissue were used as positive controls. Immunofluorescence stainings were performed according to standard histology protocols and as described before [19 (link)].
3
Western Blot Analysis of PSMA Expression
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Cell pellets were lysed on ice in Mammalian Protein Extraction Reagent (MPER, Thermo Fisher, 78,503) containing Halt Protease Inhibitor Cocktail (Thermo Fisher, 87,786). Lysates were quantified using the Pierce BCA Protein Assay kit (Thermo Fisher 23,225). 10 μg of LNCaP and 50 μg other PCA cell line samples were then denatured (95 °C for 5 minutes), separated by SDS-PAGE (4–20%), and transferred to a nitrocellulose membrane. Membranes were blocked in 5% non-fat milk, dissolved in tris-buffered saline-tween 20 (TBST), for 1 hour at room temperature prior to primary antibody incubation. PSMA protein expression was evaluated using a mouse monoclonal, anti-human PSMA antibody (Novus Biologicals, NBP1–45057) with demonstrated reactivity to canine PSMA [23 (link)]. The PSMA antibody was diluted to 1 μg/mL and membranes were incubated overnight at 4 °C, followed by secondary labelling using species a specific HRP-linked antibody (Cell Signaling) for 1 hour at room temperature. Membranes were then developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher 34,094) and ChemiDoc XRS+ molecular imager system. All membranes were re-probed for β-actin (Abcam, ab6276) for use as loading control.
4
PSMA Expression in Canine Urothelial and Prostate Cancer
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Cell lines were fixed in 10% formalin for 1 hour at room temperature. After 1-hour incubation, cells were washed and pelleted in 2% agarose, then paraffin-embedded. The University of Illinois Veterinary Diagnostic Laboratory database was reviewed for cases of iUC among female dogs (for undisputed iUC confirmation) and carcinomas of the prostate. In total, 12 iUC cases and 10 carcinomas of the prostate were identified to be recut for PSMA immunohistochemistry (IHC). IHC staining was performed using an autostainer (intelliPATH FLX, Biocare, Concord, CA). Samples were deparaffinize in xylene and rehydrated in alcohol. Samples were treated with Diva Decloaker and then blocked with Peroxidazed 1 for 5 minutes. A secondary block with Background Punisher was performed for 10 minutes. Samples were incubated with a mouse monoclonal, anti-human PSMA antibody (Novus Biologicals, NBP1–45057) at 1:1000 dilution for 30 minutes. Samples were washed, incubated with a biotinylated secondary antibody for 30 minutes, washed, then incubated with the chromogen IP FLX DAB. Slides were counterstained with hematoxylin. Well characterized human cell lines served as strong positive (LNCaP) and negative/weak positive (PC-3) PSMA controls.
5
Western Blot Analysis of PSMA Protein Expression
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Protein lysates (5–50 μg) collected from cell lines were electrophoresed on a 12% polyacrylamide gel, transferred to a nitrocellulose membrane, and block with tris buffered saline-tween 20 (TBST) with 5% nonfat dry milk (NFDM) for 1 hour at room temperature. Western blot analysis was performed using a mouse monoclonal, anti-human PSMA antibody (Novus Biological, NBP1-45057, Littleton, CO) at a concentration of 1:250 in TBST with 5% NFDM, incubated for 1 hour at room temperature. The membrane was then washed 3 times with TBST, probed with a rabbit anti-mouse secondary antibody diluted 1:5000 in TBST with 5% NFDM, and developed using ChemiDoc XRS+ molecular imager system. Band intensity analysis was done using Image Lab software. Relative protein expressions were adjusted against β-actin using anti-human antibody at a concentration of 1:5000 in TBST with 5% NFDM, incubated for 1 hour at room temperature. Results reported were derived from at least 2 independent experiments.
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