Af965

Manufactured by R&D Systems

AF965 is a laboratory product offered by R&D Systems. It is an analytical tool used for scientific research purposes. The core function of this product is to perform specific measurements or analyses, as required by the researcher's experimental protocols. No further details about the intended use or interpretation of the product's capabilities are provided.

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Lab products found in correlation

Ab24170, by Abcam (2 mentions) Ab2733, by Abcam (2 mentions) Mab1029, by R&D Systems (2 mentions) Af1515, by R&D Systems (1 mentions) Typhoon trio, by GE Healthcare (1 mentions) Percoll, by Merck Group (1 mentions) Alexa 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Infinite m1000 plate reader, by Tecan (1 mentions) Ab104224, by Abcam (1 mentions) Mabg mdect, by InvivoGen (1 mentions) Fa 11, by Bio-Rad (1 mentions) Af1029, by R&D Systems (1 mentions) Nb100 1028, by Novus Biologicals (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Af1443, by R&D Systems (1 mentions) Zen 2.1 blue edition software, by Zeiss (1 mentions) A0564, by Agilent Technologies (1 mentions) Nb300 575, by Novus Biologicals (1 mentions) Tgn38, by Novus Biologicals (1 mentions) A5441, by Merck Group (1 mentions)

7 protocols using af965

Quantifying Cathepsin Activity in Neuroblastoma Cells

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SH-SY5Y and SK-N-BE2 neuroblastoma cells were seeded in equal density in 6-well plates. When cells reached 80% confluency, 0.1 μM BMV109 pan cathepsin activity-based probe [51 (link)] was added to the cell and incubated for 1 h. Following incubation cells were washed thrice with PBS. Cells were then harvested by scraping and lysed for analysis. Lysis buffer consisted of 50 mM citrate, pH 5.5, 0.5% CHAPS, 0.1% Triton X-100 and 4 mM DTT. The lysates were quantified using BCA assay and equal amount of sample (30 μg) was analysed on a gel. For cathepsin analysis, 30 μg of total protein from each secretome sample was taken and BMV109 probe (0.1 μM) was added to each sample and incubated at 37°C for 1 h. At the end of the incubation, SDS sample loading buffer was added to each sample and analysed on a gel. The Cy5 fluorescence of the probe was detected by scanning the gels under the Typhoon Trio (GE Healthcare) scanner. The gels was then transferred to a nitrocellulose membrane and probed with antibodies against Cathepsin isoforms B and L (R&D AF1515 and AF965 antibodies, respectively). The specificity of the probe was tested by growing cells in the presence of the cysteine cathepsin inhibitor JPM (100 μM) for 24 h and then incubating with the probe.

Gangoda L., Keerthikumar S., Fonseka P., Edgington L.E., Ang C.S., Ozcitti C., Bogyo M., Parker B.S, & Mathivanan S. (2015). Inhibition of cathepsin proteases attenuates migration and sensitizes aggressive N-Myc amplified human neuroblastoma cells to doxorubicin. Oncotarget, 6(13), 11175-11190.

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Subcellular Fractionation of Rat Cortical Neurons

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Rat cortical neurons in DIV7 were homogenized in homogenization buffer (250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, and protease inhibitor mixture). After removing the unbroken cells and nuclei by centrifugation at 750 g for 10 min at 4°C, the supernatant (one volume) was laid on the top of nine volumes of 20% (vol/vol) Percoll (GE17-0891; Sigma-Aldrich) with homogenization buffer and centrifuged at 20,000 g for 2.5 h at 4°C. 14 fractions were sequentially collected from top (fraction 1) to bottom (fraction 14). Equal amounts of each fraction were analyzed by sequential immunoblotting with antibodies against LAMP1 (ab24170; Abcam), LAMP2 (NB300-591; Novus Biologicals), cathepsin B (AF965; R&D Systems), cathepsin D (MAB1029; R&D Systems), EEA1 (610456; BD), Rab7 (9367P; Cell Signaling Technology), and M6PR (ab2733; Abcam). The blot membranes were stripped between applications of each antibody.

Cheng X.T., Xie Y.X., Zhou B., Huang N., Farfel-Becker T, & Sheng Z.H. (2018). Characterization of LAMP1-labeled nondegradative lysosomal and endocytic compartments in neurons. The Journal of Cell Biology, 217(9), 3127-3139.

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Cell Surface Cathepsin B Quantification

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Cells (40,000 per well) were seeded onto black 96-well plates with transparent bottoms and grown overnight. Active endocytosis was initially blocked by a 1 h incubation at 4 °C; to prevent any additional endocytosis, all subsequent steps were also performed at 4 °C. Goat anti-mouse CatB polyclonal antibodies (1:1000, AF965, R&D), mouse anti-human CatB monoclonal antibodies 3E1 (100 nM, 6 (link)) or biotinylated DARPins (100 nM) were next added in PBS supplemented with 3 % BSA and incubated for 1 hour. After washing with PBS, the Alexa488-conjugated secondary anti-goat antibodies (1:1000, Thermo Fisher Scientific), anti-mouse antibodies (1:1000, Thermo Fisher Scientific) or Alexa488-conjugated streptavidin (1:1000, Thermo Fisher Scientific) was added for 1 hour. After washing, the cell membrane-associated fluorescence (ex/em = 488/520 nm) was monitored in a Tecan Infinite M-1000 plate reader by measuring nine locations in each well. The mean background fluorescence (where only secondary antibody or only streptavidin was incubated) was subtracted from the measurements. In the control experiments in which unlabelled competitor DARPins were used to block cell membrane-associated CatB binding to biotinylated DARPin 8h6, the competitor DARPins (1 μM) were incubated with cells during the last 30 min of active endocytosis blocking.

Kramer L., Renko M., Završnik J., Turk D., Seeger M.A., Vasiljeva O., Grütter M.G., Turk V, & Turk B. (2017). Non-invasive in vivo imaging of tumour-associated cathepsin B by a highly selective inhibitory DARPin. Theranostics, 7(11), 2806-2821.

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Immunohistochemistry of Microglia and Astrocytes

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30μm microtome-cut sagittal sections were incubated in X34 for one hour at room temperature in 60% PBS/40% EtOH mix; pH was adjusted with 1N NaOH. Following this, sections were washed briefly with 60% PBS/40% EtOH before blocking in 5% donkey or goat serum for 1h at room temperature. Primary antibodies were diluted in blocking buffer and incubated at 4°C overnight with slow agitation unless mentioned otherwise. Primary antibodies were used as follows: IBA1 (1:500; 019-19741, Fujifilm or NB100-1028, Novus Biologicals), CD68 (1:100; FA-11, BioRad), P2RY12 (1:100 at room temperature, S16007D, BioLegend), TMEM119 (1:500; E3E1O, Cell Signaling Technology), Clec7a (1:50 at room temperature; mabg-mdect, InvivoGen), GFAP (1:2000; 2E1.E9 Alexa Flour 488-conjugated, BioLegend), 6E10 (1:1000; Alexa Flour 647-conjugated, BioLegend), NeuN (1:2000; ab104224, Abcam), LAMP1/CD107a (1:500; AF4320, Novus Biologicals), CathepsinB (1:50; AF965, R&D Systems), Cathepsin D (1:50; AF1029, R&D Systems). The next day, sections were washed and incubated with secondary antibody diluted in blocking buffer, followed by a 4′,6-Diamidin-2-phenylindol (DAPI, 5μg/mL) where appropriate before mounting sections onto slides (Prolong^™ Gold Antifade reagent, Thermo Fisher Scientific).

Sharma M., Ellis R.L, & Hinton D.M. (1992). Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage.. Proceedings of the National Academy of Sciences of the United States of America, 89(14), 6658-6662.

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Immunofluorescence Staining of Cathepsin B and FABP Proteins

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Sections of 20 μm thickness were stained in immunofluorescence with goat anti-human cathepsin B (AF965; 10 μg/ml, R&D Systems) or anti-mouse FABP4 (AF1443; 10 μg/ml, R&D Systems) and Cy3-conjugated donkey anti-goat IgG (AP180C; 1:200, Merck Millipore), or rabbit anti-rat FABP5 (0.5 μg/ml, Owada et al. 2001 (link)) and Cy3-conjugated donkey anti-rabbit IgG (AP182C; 1:200; Merck Millipore). Sections were subsequently treated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, ImmunoBioScience) for nuclear staining. 3D images and movies were reconstructed by use of Zen 2.1 [blue edition] software (Carl Zeiss).

Bando Y., Tokuda N., Ogasawara Y., Onozawa G., Nagasaka A., Sakiyama K., Owada Y, & Amano O. (2021). Expression and enhancement of FABP4 in septoclasts of the growth plate in FABP5-deficient mouse tibiae. Histochemistry and Cell Biology, 155(4), 439-449.

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Immunofluorescence and Western Blotting Antibodies

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Antibodies used for immunofluorescence were as follows: Insulin (MA1-10517; Thermo Fischer Scientific and A0564; Dako), CGB (259 10; Synaptic systems), CGA (NB120-15160; Novus biologicals), TGN 38 (NB300-575; Novus biologicals and 610898; BD Biosciences), CPE (13710-1-AP; Proteintech) M6PR (16795-1-AP; Proteintech), IGF2R (20253-1-AP; Proteintech), SCG III (10954-1-AP; Proteintech), PCSK2 (PA1-058; Invitrogen), HA (11867423001; Roche/Sigma-Aldrich), Cathepsin B (AF965; R&D systems).
For purposes of western blotting, antibodies against GFP (11814460001; Roche/Sigma-Aldrich) SNAP-tag (P9310S; NEB), and β-actin (A5441; Sigma-Aldrich) were used.

Parchure A., Tian M., Stalder D., Boyer C.K., Bearrows S.C., Rohli K.E., Zhang J., Rivera-Molina F., Ramazanov B.R., Mahata S.K., Wang Y., Stephens S.B., Gershlick D.C, & von Blume J. (2022). Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules. The Journal of Cell Biology, 221(12), e202206132.

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Multimodal Lysosomal Imaging and Manipulation

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Sources of antibodies or reagents are as follows: antibodies against LAMP1 (rabbit; ab24170; Abcam; and rat; 1D4B; Developmental Studies Hybridoma Bank), cathepsin D (rat; MAB1029; R&D Systems), cathepsin B (goat; AF965; R&D Systems), βIII-tubulin (mouse; MAB5564; EMD Millipore; and mouse; T8578; Sigma-Aldrich), MAP2 (rabbit; AB5622; EMD Millipore; and mouse; 556320; BD), Tau-1 (mouse; MAB3420; EMD Millipore), NeuN (mouse; MAB377; EMD Millipore), GBA (rabbit; G4171; Sigma-Aldrich), CI-M6PR (mouse; ab2733; Abcam), EEA1 (mouse; 610456; BD), Rab7 (rabbit; 9367P; Cell Signaling Technology), Rab9a (mouse; MA3-067; Thermo Fisher Scientific); BSA-gold tracer (6 nm; Electron Microscopy Sciences); BODIPY FL–pepstatin A (Invitrogen); Alexa Fluor 546– or 633–conjugated secondary antibodies (Invitrogen), and Bouin’s solution (HT10132; Sigma-Aldrich). LAMP1-mApple (promoter, phosphoglycerate kinase; backbone, pLex; lentiviral) was generated in M. Ward’s laboratory (National Institute of Neurological Disorders and Stroke, Bethesda, MD). mRFP-Rab7 (promoter, cytomegalovirus; backbone, pmRFP-C3) was a gift from A. Helenius (14436; Addgene; Vonderheit and Helenius, 2005 (link)). The constructs were verified by DNA sequencing. Stealth siRNAs are from Thermo Fisher Scientific (LAMP1-siRNA1, MSS237022; LAMP1-siRNA2, MSS275304; cathepsin D–siRNA1, MSS203352; and cathepsin D–siRNA2, MSS203353).

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