Haf007

Manufactured by R&D Systems

Sourced in United States

The HAF007 is a laboratory equipment product offered by R&D Systems. It is a device designed for a specific core function, but a detailed description while maintaining an unbiased and factual approach is not available.

Automatically generated - may contain errors

Lab products found in correlation

Haf008, by R&D Systems (11 mentions) Ab16284, by R&D Systems (3 mentions) Sc 166029, by Abcam (3 mentions) Haf016, by R&D Systems (3 mentions) Sc 47778, by Santa Cruz Biotechnology (3 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Anti klf4 primary antibody sc 20691, by Santa Cruz Biotechnology (2 mentions) Ab133267, by Abcam (2 mentions) Ripa buffer, by Cell Signaling Technology (2 mentions) Azure 300, by Azure Biosystems (2 mentions) Ab182561, by Cell Signaling Technology (2 mentions) Sc 32761, by Santa Cruz Biotechnology (2 mentions) Sc 166944, by Santa Cruz Biotechnology (2 mentions) Haf005, by R&D Systems (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Mab8929, by R&D Systems (2 mentions) Mab375, by R&D Systems (2 mentions) Qubit protein assay, by Thermo Fisher Scientific (1 mentions) Ab129002, by Abcam (1 mentions) West q pico ecl solution, by GenDEPOT (1 mentions)

Spelling variants of this product

Goat anti-mouse HRP-conjugated (HAF007), (2 citations) Anti-mouse IgG (HAF007 (2 citations)

34 protocols using haf007

Localization and Purification of Mycobacterial Proteins

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Plasmids required for the localization of GFP (pDMNI) and EccA₃-GFP (pDMNI0615) were transformed into WT_MS and ΔESX-3_MS to generate strains described in Table S2. Protein expression was confirmed through immunoblotting using a mouse derived anti-GFP antibody (F56-6A1, Santa Cruz) and a goat anti-mouse horseradish peroxidase conjugated antibody (HAF007, R&D systems) (Fig. S1). The primary and secondary antibodies were used at the final titres of 1:2000 and 1:10,000, respectively.
Plasmids constructed for the purification of FLAG (pNFLAG) and FLAG-MSMEG_0615 (pNFLAG0615) were transformed into WT_MS for the purification of possible interacting proteins to generate strains described in Table S2. Protein expression was confirmed using immunoblotting with a mouse derived anti-FLAG antibody (FG4R, ThemoFisher Scientific) and a goat anti-mouse horseradish peroxidase conjugated antibody (HAF007, R&D systems) (Fig. S2). The FG4R antibody was used at a final titre of 1:5000.

Kriel N.L., Newton-Foot M., Bennion O.T., Aldridge B.B., Mehaffy C., Belisle J.T., Walzl G., Warren R.M., Sampson S.L, & Gey van Pittius N.C. (2022). Localization of EccA3 at the growing pole in Mycobacterium smegmatis. BMC Microbiology, 22, 140.

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Exosomal Protein Profiling by Western Blot

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Cell lysate was collected in 8 M urea with 2.5% SDS. Protein concentration was measured with Qubit Protein Assay (Thermo Fisher), with 30 μg of cell lysate and 15 μg of exosome protein loaded. Protein was denatured in 4× NuPAGE LDS sample buffer with 62.5 mM DTT for 10 min at 70 °C. Membranes were blocked with 5% milk in TBS with 0.1% Tween (TBST) and incubated with rabbit anti-SLC19A1 (Boster Biological Technology PB9504, 1:1000), rabbit anti-SLC38A2 (Sigma Aldrich SAB4502246, 1:1000), mouse anti-CD81 (Santa Cruz Biotechnology sc-166029, 1:1000), or rabbit anti-vinculin (Abcam ab129002, 1:1000) for 1 h at room temperature or overnight at 4 °C. Membranes were incubated in anti-rabbit HRP (Abcam ab16284, 1:1000) or anti-mouse HRP (R&D Systems HAF007, 1:1000) for 1 h at room temperature and developed using West-Q Pico ECL solution (GenDEPOT).

McAndrews K.M., Che S.P., LeBleu V.S, & Kalluri R. (2021). Effective delivery of STING agonist using exosomes suppresses tumor growth and enhances antitumor immunity. The Journal of Biological Chemistry, 296, 100523.

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Western Blot Analysis of KLF4 Expression

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Western blot was performed as described (10 (link)). Anti-KLF4 primary antibody (sc-20691; dilution 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) was used with GAPDH (5174; dilution 1:10000; Cell Signaling Technologies, Danvers, MA, USA) or ACTIN (A5441; dilution 1:10000; Sigma-Aldrich) as a loading control. Membranes were incubated with rabbit or mouse HRP-conjugated secondary antibodies (HAF008 and HAF007; dilutions range 1:5000–1:20000; R&D Systems, Minneapolis, MN, USA).

Tassone E., Bradaschia-Correa V., Xiong X., Sastre-Perona A., Josephson A.M., Khodadadi-Jamayran A., Melamed J., Bu L., Kahler D.J., Ossowski L., Leucht P., Schober M, & Wilson E.L. (2019). KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone. Oncogene, 38(29), 5766-5777.

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Hypoxia and Gemcitabine Modulate Extracellular Vesicles

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MIA PaCa-2 cells (triplicates) were exposed to hypoxia (1%O₂) /normoxia (21% O₂) or to gemcitabine (1μM)/vehicle (cell culture grade water) for 72h and cultured in RPMI medium supplemented with EVs-depleted FBS. EVs were collected as previously described and pellets were resuspended in 100uL of PBS 1x. For western blot, 20uL of EVs preparation from each condition was loaded into the gel. CD81 (1;5000 Santa Cruz Cat# sc-166029, RRID:AB_2275892), Syntenin-1 (1:5000, Abcam Cat# ab133267, RRID:AB_11160262) and CD9 (1:1000, Abcam Cat# ab263019), were used to detect EVs.
Anti-Rabbit-HRP 1:5000 (CST Cat#7074S, RRID:AB_2099233) was used for Syntenin-1, Anti-Rabbit-HRP 1:2000 (Abcam, Cat# ab16284, RRID:AB_955387) was for CD9 and anti-mouse-HRP (1:2000 R&D Cat# HAF007, RRID:AB_357234) for CD81.

Ruivo C.F., Bastos N., Adem B., Batista I., Duraes C., Melo C.A., Castaldo S.A., Campos‐Laborie F., Moutinho-Ribeiro P., Morão B., Costa-Pinto A., Silva S., Osorio H., Ciordia S., Costa J.L., Goodrich D., Cavadas B., Pereira L., Kouzarides T., Macedo G., Maio R., Carneiro F., Cravo M., Kalluri R., Machado J.C, & Melo S.A. (2022). Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression. Gut, 71(10), 2043-2068.

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Arid1a Regulation in Dental Progenitor Cells

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At PN7.5,4 days after tamoxifen induction, the apical thirds of first mandibular molars from Gli1-CreER;Arid1a^fl/fl mice and littermate controls were cut into pieces and homogenized in RIPA buffer (Cell Signaling, 9806s) supplemented with protease inhibitor (Thermo Fisher Scientific, A32959). Western blot was performed per standard protocol and signals were detected using Azure 300 (Azure biosystems). The primary antibodies are listed in Table S1. HRP-conjugated secondary antibodies (R&D, HAF007, HAF008, and HAF016) were used in the study. For co-immunoprecipitation (co-IP), DPCs cultured in vitro were harvested and lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA (pH 8.0), 1mM PMSF, 1% NP-40, 5% glycerol]. Then lysates were subjected to immunoprecipitation with anti-Arid1a antibody (Abcam, ab182561) or normal Rabbit IgG (Cell Signaling Technology, 2729) and protein A-Sepharose (VWR, CA97067-898). Immune complexes were washed and subjected to immunoblotting with anti-Arid1a (Santa Cruz, sc-32761) or anti-Plagl1 (Santa Cruz, sc-166944) antibodies.

Du J., Jing J., Yuan Y., Feng J., Han X., Chen S., Li X., Peng W., Xu J., Ho T.V., Jiang X, & Chai Y. (2021). Arid1a-Plagl1-Hh signaling is indispensable for differentiation-associated cell cycle arrest of tooth root progenitors. Cell reports, 35(1), 108964.

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Immunohistochemistry and RNA in situ Hybridization of Inflammatory Markers

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For immunohistochemistry (IHC), sectioned formalin-fixed paraffin-embedded biopsy samples were deparaffinized and rehydrated, which was followed by heat-induced and enzymatic antigen retrieval. Subsequently, sections were blocked and incubated at 4 °C overnight with the following primary antibodies: anti-IL-1β antibody (1:100, 12242; Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-IL-18 antibody (1:1000, ab243091; Abcam, Cambridge, UK). Thereafter, biotinated or horseradish peroxidase-conjugated secondary antibodies (HAF007; R&D Systems, Inc., Minneapolis, MN, USA) were used for detection. Anti-CD66b antibodies (1:100, 3929023, Biolegend, San Diego, CA, USA) were used for the detection of neutrophils. Images were captured using Axio Scope AI (ZEISS, Oberkochen, Germany).
For RNA in situ hybridization, the RNAScope Kit (Advanced Cell Diagnostics, Newark, CA, USA) was used to measure the mRNA expression in paraffin-embedded sections.

Furuta Y., Gushima R., Naoe H., Honda M., Tsuruta Y., Nagaoka K., Watanabe T., Tateyama M., Fujimoto N., Hirata S., Miyagawa E., Sakata K., Mizuhashi Y., Iwakura M., Murai M., Matsuoka M., Komohara Y, & Tanaka Y. (2023). Possible Association of Mutations in the MEFV Gene with the Intestinal Phenotype of Behçet’s Disease and Refractoriness to Treatment. Journal of Clinical Medicine, 12(9), 3131.

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Quantifying Cellular Protein Levels by Western Blot

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Protein levels of proCPA1 and immunoglobulin binding protein (BiP) were evaluated by Western blotting in total, soluble, and insoluble fractions of cell lysates. Ten µL of each fraction was analyzed per lane. As a loading control, human α-tubulin was measured. Incubation with the primary and secondary antibodies was performed overnight at 4 °C and for 1 h at room temperature, respectively. Protein bands were detected using the SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Thermo Scientific, Waltham, MA, USA). Rat monoclonal antibody against human carboxypeptidase A1 (MAB2856, R&D Systems Minneapolis, MN, USA) was used at 1:500 dilution. Mouse monoclonal antibody (DM1A) against α-tubulin (CP06, MilliporeSigma, St. Louis, MO, USA) was used at 1:2000 dilution. BiP was detected by a rabbit polyclonal anti-GRP78 antibody (ab21685, Abcam, Cambridge, MA, USA) used at 1:2000 dilution. Horse-radish peroxidase (HRP)-conjugated goat polyclonal anti-rat IgG (HAF005, R&D Systems, Minneapolis, MN, USA) was used at 1:1000 dilution. HRP-conjugated goat polyclonal anti-mouse IgG (HAF007, R&D Systems) was used at 1:2500 dilution. HRP-conjugated goat polyclonal anti-rabbit IgG (HAF008, R&D Systems) was used at 1:2000 dilution.

Sándor M., Thiel F.G., Schmid M., Demcsák A., Morales Granda N.C., Németh B.C., Vajda S., Hoerning A, & Sahin-Tóth M. (2022). Novel p.G250A Mutation Associated with Chronic Pancreatitis Highlights Misfolding-Prone Region in Carboxypeptidase A1 (CPA1). International Journal of Molecular Sciences, 23(24), 15463.

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Subcellular Protein Isolation and Western Blot Analysis

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Nuclear, cytoplasmic (CYT) and plasma membrane (PM) protein of PFC tissues or astrocytes were isolated by commercial kits (KGP1100, KeyGEN BioTECH; P0033, Beyotime Biotechnology). Phenylmethanesulfonyl fluoride (PMSF, a protease inhibitor) was used to inhibit protein degradation. The protein concentration of sample was detected by bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Lysates were loaded into 10% sodium dodecyl sulfate-polyacrylamid gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% non-fat milk for 1 h. Then, the following first antibodies were used: GLUT1 (1:5000, ab115730, Abcam), TXNIP (1:1000, #14715, CST), Na, K ATPase (1:1000, #3010, CST), GR (1:1000, #3600, CST), Histone H3 (1:500, sc-517576, Santa Cruz) and β-actin (1:2000, #4970, CST). After incubating with first antibody overnight, membranes were incubated goat anti-mouse IgG (1:5000, HAF007, R&D systems) or goat anti-rabbit IgG (1:3000, #7074, CST) for 1.5 h. Protein bands were visualized by ECL (#180–5001, Tanon) and imaged by chemiluminescence imaging instrument (#5200, Tanon). The gray value of images was measured by ImageJ.

Pan S.M., Zhou Y.F., Zuo N., Jiao R.Q., Kong L.D, & Pan Y. (2022). Fluoxetine increases astrocytic glucose uptake and glycolysis in corticosterone-induced depression through restricting GR-TXNIP-GLUT1 Pathway. Frontiers in Pharmacology, 13, 872375.

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Western Blot Analysis of Apoptosis-Related Proteins

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Mouse monoclonal antibody raised against Bcl-2 (#15071) and rabbit monoclonal antibody against Bcl-xL (#2764) were obtained from Cell Signaling Technology (Danvers, MA). Rabbit polyclonal antibody against the D2 receptor (MBS612859) was purchased from MyBioSource (San Diego, CA) and rabbit polyclonal antibody against the B2 receptor (NBP2-14351) from Novus Biologicals (Littleton, CO). Mouse monoclonal antibodies against β-actin (MAB8929) and Bax (MAB846) as well as goat polyclonal antibody against mouse or rabbit IgG conjugated with horseradish peroxidase (HAF007 or HAF008, respectively) were supplied by R&D Systems (Minneapolis, MN). Mouse monoclonal antibody against endothelial nitric oxide synthase (NOS3) (sc-376751) and against phospho-NOS3 (pNOS3) (sc-293032) were purchased from Santa Cruz Biotechnology (Dallas, TX). Goat polyclonal antibody against rabbit IgG conjugated with Alexa-Fluor488 (A11008) was obtained from Thermo Fisher Scientific.

Niewiarowska-Sendo A., Kozik A, & Guevara-Lora I. (2018). Influence of bradykinin B2 receptor and dopamine D2 receptor on the oxidative stress, inflammatory response, and apoptotic process in human endothelial cells. PLoS ONE, 13(11), e0206443.

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Immunoprecipitation and Western Blotting

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Anti-HA agarose beads (ThermoFisher 26181) were incubated with cell supernatant or mouse ascites for 2 h at 4 °C on a nutator. The beads were washed 2x with cold PBS, and Laemmli sample buffer (BioRad 161–0747) with or without β-mercaptoethanol (BME) was added. Protein samples (immunoprecipitate or total cell lysates) were homogenized and heated 3x for 3 min at 95 °C and resolved by SDS-PAGE. Gels were transferred to nitrocellulose membranes and blotted for respective antibodies in TBST (ThermoFisher 28360). Detection of antibody was achieved with Pierce ECL femto western substrate (ThermoFisher 34095). The following antibodies were used for immunoblot: mouse IgG HRP-conjugated antibody (R&D systems HAF007), rabbit IgG HRP-conjugated antibody (R&D systems HAF008), anti-HA (Invitrogen 26183). Polyclonal anti-CPG2 and anti-β-Lac antibodies were raised in rabbits by inoculation with whole recombinant protein produced in E. coli and purified by nickel bead affinity chromatography (service performed by GenScript).

Gardner T.J., Lee J.P., Bourne C.M., Wijewarnasuriya D., Kinarivala N., Kurtz K.G., Corless B.C., Dacek M.M., Chang A.Y., Mo G., Nguyen K.M., Brentjens R.J., Tan D.S, & Scheinberg D.A. (2021). Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ. Nature chemical biology, 18(2), 216-225.

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