Ab99697

Manufactured by Abcam

Sourced in China, United States

Ab99697 is a monoclonal antibody produced in mouse. It is designed for use in various research applications.

Automatically generated - may contain errors

Lab products found in correlation

Cst 58 802 s, by Cell Signaling Technology (3 mentions) Ab9485, by Abcam (3 mentions) Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (2 mentions) Ab48394, by Abcam (2 mentions) Radioimmunoprecipitation assay lysis buffer, by Thermo Fisher Scientific (2 mentions) Image pro plus software 6, by Media Cybernetics (2 mentions) Ab97051, by Abcam (2 mentions) Ab76252, by Abcam (1 mentions) Ab99702, by Abcam (1 mentions) Ab126622, by Abcam (1 mentions) Ab52771, by Abcam (1 mentions) P yap, by Abcam (1 mentions) Gapdh bsm 33033m, by Bioss Antibodies (1 mentions) A25012, by Abbkine (1 mentions) C myc, by Abcam (1 mentions) Ab32072, by Abcam (1 mentions) Mg 63, by National Collection of Authenticated Cell Cultures (1 mentions) Saos 2, by National Collection of Authenticated Cell Cultures (1 mentions) Mnng hos, by National Collection of Authenticated Cell Cultures (1 mentions) Ab125248, by Abcam (1 mentions)

17 protocols using ab99697

Osteosarcoma Cell Lines and Antibody Specifications

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The human osteosarcoma cell lines MG63, MNNG-HOS and Saos-2 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human osteosarcoma cell line U-2OS was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were cultured following the ATCC protocols. Standardized culture conditions had been described previously [18 (link)].
The antibodies used were S1PR3 (ab126622; Abcam, Cambridge, UK), YAP (ab52771; Abcam, Cambridge, UK), p-YAP (ab76252; Abcam, Cambridge, UK), c-Myc (ab32072; Abcam), Ki67 (GB13030; Servicebio, Wuhan, China), β-actin (M1210-2; Hua'an Biology, Chuzhou, China), GAPDH (bsm-33033M; Bioss, Beijing, China), anti-rabbit IgG light chain (ab99697, Abcam), anti-rabbit IgG heavy chain (ab99702, Abcam), and anti-mouse IgG light chain (A25012, Abbkine, CA, USA).

Shen Y., Zhao S., Wang S., Pan X., Zhang Y., Xu J., Jiang Y., Li H., Zhang Q., Gao J., Yang Q., Zhou Y., Jiang S., Yang H., Zhang Z., Zhang R., Li J, & Zhou D. (2018). S1P/S1PR3 axis promotes aerobic glycolysis by YAP/c-MYC/PGAM1 axis in osteosarcoma. EBioMedicine, 40, 210-223.

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Quantification of PKM2 and P-PKM2 in Murine Retina

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Following the protocol described by Tsang et al. (1998 (link), 1996 (link)), retina samples were collected from mice at five weeks (P36-P39). To evaluate PKM2 expression levels, the samples were homogenized using a solution consisting of M-PER Mammalian Protein Extraction Reagent (Prod #78501 Thermo Scientific), phosphatase inhibitor cocktail (catalog P2850–5 mL; Sigma), and protease inhibitor cocktail (catalog P8340–1 mL; Sigma). After transfer onto nitrocellulose membrane (BioRad), samples were blocked in 5% skim milk 902887 MP Biomedicals, LLC). The membrane was then immersed overnight at 4 °C in the following primary antibodies: rabbit Anti-PKM2 (1:1000; #3198S; Cell Signaling Technology), mouse anti–β-actin (1:1000; ab125248; Abcam), and rabbit anti-phospho-PKM2 (1:1000; #3827S; Cell Signaling Technology). The following day, the membrane was washed in 0.5% PBST (500 μl Tween-20 in 1000 ml PBS), and subsequently incubated in mouse monoclonal anti-rabbit IgG-HRP antibody (1:2000; ab99697; Abcam) and rabbit anti-mouse IgG-HRP antibody (1:2,000; sc-358914; Santa Cruz Biotechnology Inc.). Chemiluminescence (EMD Millipore) was detected using Biomax film (Kodak), and PKM2, P-PKM2, and actin levels were then quantified.

Zhang E., Ryu J., Levi S.R., Oh J.K., Hsu C.W., Cui X., Lee T.T., Wang N.K., de Carvalho J.R, & Tsang S.H. (2020). PKM2 ablation enhanced retinal function and survival in a preclinical model of retinitis pigmentosa. Mammalian genome : official journal of the International Mammalian Genome Society, 31(3-4), 77-85.

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Co-immunoprecipitation Assay Protocol

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For the co-IP assay, the SH-SY5Y cells were harvested 48 h post-transfection and lysed for 20 min in IP lysis buffer (25 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1 mM EDTA, 1% NP-40, 5% glycerol, 1 mM PMSF, and protease inhibitor cocktail), and the amount of protein was estimated using Bradford reagent. Then, 2–3 mg of the cell lysates were immunoprecipitated with the respective antibodies at 4 °C overnight and incubated with 25 µL of protein agarose beads at 4 °C for 3 h. Before loading the samples on the SDS-PAGE gels, the beads were washed with lysis buffer and eluted in 2X sodium dodecyl sulfate (SDS) sample loading buffer (5X SDS sample loading buffer containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris-HCl [pH 6]). The samples were then boiled at 95–100 °C for 5 min and analyzed by western blotting. Mouse IgG (ab-99,697, 1:10000, Abcam) and rabbit IgG (CST-58,802 S, 1:10000, Cell Signaling Technology) light chain-specific secondary antibody was used to prevent interference from heavy and light immunoglobulin chains for the binding assay.

Karapurkar J.K., Kim M.S., Colaco J.C., Suresh B., Sarodaya N., Kim D.H., Park C.H., Hong S.H., Kim K.S, & Ramakrishna S. (2023). CRISPR/Cas9-based genome-wide screening of the deubiquitinase subfamily identifies USP3 as a protein stabilizer of REST blocking neuronal differentiation and promotes neuroblastoma tumorigenesis. Journal of Experimental & Clinical Cancer Research : CR, 42, 121.

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Protein Extraction and Western Blot Analysis

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Cells were lysed with RIPA buffer (Sigma-Aldrich) with added protease inhibitors (Sigma-Aldrich). Proteins were obtained by centrifugation at 12,500 rpm at 4 °C for 30 min and quantified using an EZQ Protein Quantitation Kit (Life Technologies) according to manufacturer’s instructions. Proteins (30 µg) were separated by electrophoresis on SDS-PAGE (Bio-Rad). SeeBlue Plus2 (Invirogen) was used for sizing and visualization of the gel running pattern and protein transfer. Resolved proteins were transferred on immobilon PVDF membranes (Millipore) at 20 V for 30 min. Membranes were air-dried, wet with methanol, washed with TBS-T and blocked at 4 °C overnight in 1% (w/v) non-fat skimmed milk/TBS-T. Membranes were incubated with antibodies against AR (1:500 ab108341), GABBR1 (1:500 ab75239) from ABCAM, GAPDH (1:20000 5174 s), ENO2 (1:200 9536 s) obtained from Cell Signaling and GRP (1:500 sc-271045) from Santa Cruz Biotechnology at 4 °C overnight.
Blots were then incubated with anti-rabbit secondary antibody (1:5000 ab99697) obtained from ABCAM or anti-mouse secondary antibody (1:300 7076 s) from Cell Signaling conjugated to horseradish peroxidase (HRP) at room temperature (RT) for 2 h. Chemiluminescence signals were detected using ChemiDoc XRS system (BioRad). Western immunoblots were quantified using ImageJ software.

Solorzano S.R., Imaz-Rosshandler I., Camacho-Arroyo I., García-Tobilla P., Morales-Montor G., Salazar P., Arena-Ortiz M.L, & Rodríguez-Dorantes M. (2018). GABA promotes gastrin-releasing peptide secretion in NE/NE-like cells: Contribution to prostate cancer progression. Scientific Reports, 8, 10272.

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TGFβR2 Protein Expression Analysis

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C666-1 cells were lysed in cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). Protein concentration was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein (50 µg) was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). Subsequently, the membrane was blocked in 5% non-fat dried milk in Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific, Inc.) for 3 h at room temperature. The PVDF membrane was then incubated with rabbit anti-TGFβR2 monoclonal antibody (1:500; ab184948; Abcam, Cambridge, MA, USA), or rabbit anti-GAPDH monoclonal antibody (1:250; ab181602; Abcam) as an internal reference, for 3 h at room temperature. Subsequently, the membrane was washed with DPBS for 10 min and then incubated with mouse anti-rabbit secondary antibody (1:5,000; ab99697; Abcam) for 1 h at room temperature. Following washing with DPBS for 15 min, the immune complexes on the PVDF membrane were detected using an enhanced chemiluminescence western blotting kit (Pierce; Thermo Fisher Scientific, Inc.). Image-Pro Plus v. 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze relative protein expression levels, represented as the density ratio vs. GAPDH.

Ma F., Wang Z., Wang J., Liu X, & Hu C. (2017). MicroRNA-19a promotes nasopharyngeal carcinoma by targeting transforming growth factor β receptor 2. Experimental and Therapeutic Medicine, 14(2), 1419-1426.

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Immunoprecipitation and Western Blotting

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Cells were transfected with the indicated DNA constructs. At 36–48 h post transfection, the cells were lysed in IP lysis buffer ((25 mM Tris–HCl (pH 7.4), 150 mM sodium chloride, 1 mM EDTA, 1% NP-40, 5% glycerol, 1 mM PMSF, and protease inhibitor cocktail) for 20 min and the amount of protein was estimated using Bradford reagent. Cell lysate (2–3 mg) was immunoprecipitated using the indicated antibodies at 4 °C overnight and then incubated with 35 μL of protein agarose beads at 4 °C for 3 h. The agarose beads were washed with lysis buffer and eluted in 2X SDS sample loading buffer (5X SDS sample loading buffer containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris–HCl [pH 6]). The eluted samples were boiled at 95 °C–100 °C for 5 min and separated on SDS-PAGE gels by western blotting. Mouse IgG (ab-99697, 1: 10,000; Abcam) and rabbit IgG (CST-58802S, 1: 10,000; Cell Signaling Technology) light chain-specific secondary antibody was used to prevent interference from heavy and light immunoglobulin chains in the binding assay.

Karapurkar J.K., Colaco J.C., Suresh B., Tyagi A., Woo S.H., Jo W.J., Ko N., Singh V., Hong S.H., Oh S.J., Kim K.S, & Ramakrishna S. (2024). USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells. Cellular and Molecular Life Sciences: CMLS, 81(1), 145.

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Histone Variant Detection by Western Blot

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Isolated histones were separated on a 4 to 12% NuPAGE bis-tris gel and transferred to a 0.2-μm nitrocellulose blotting membrane. Membranes were blocked in TBS-T [50 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] containing 5% milk powder and probed for H3.3 (Thermo Fisher Scientific, MA5-24667; 1:5000), H3 (Abcam, ab1791; 1:5000), and H4 (Abcam, ab7311; 1:5000) primary antibodies diluted in TBS-T containing 5% milk powder. This was followed by anti-rabbit horseradish peroxidase (Abcam, ab99697; 1:10,000) and visualized using enhanced chemiluminesence prime Western blotting detection chemiluminescent reagent.

Bryant L., Li D., Cox S.G., Marchione D., Joiner E.F., Wilson K., Janssen K., Lee P., March M.E., Nair D., Sherr E., Fregeau B., Wierenga K.J., Wadley A., Mancini G.M., Powell-Hamilton N., van de Kamp J., Grebe T., Dean J., Ross A., Crawford H.P., Powis Z., Cho M.T., Willing M.C., Manwaring L., Schot R., Nava C., Afenjar A., Lessel D., Wagner M., Klopstock T., Winkelmann J., Catarino C.B., Retterer K., Schuette J.L., Innis J.W., Pizzino A., Lüttgen S., Denecke J., Strom T.M., Monaghan K.G., Yuan Z.F., Dubbs H., Bend R., Lee J.A., Lyons M.J., Hoefele J., Günthner R., Reutter H., Keren B., Radtke K., Sherbini O., Mrokse C., Helbig K.L., Odent S., Cogne B., Mercier S., Bezieau S., Besnard T., Kury S., Redon R., Reinson K., Wojcik M.H., Õunap K., Ilves P., Innes A.M., Kernohan K.D., Costain G., Meyn M.S., Chitayat D., Zackai E., Lehman A., Kitson H., Martin M.G., Martinez-Agosto J.A., Nelson S.F., Palmer C.G., Papp J.C., Parker N.H., Sinsheimer J.S., Vilain E., Wan J., Yoon A.J., Zheng A., Brimble E., Ferrero G.B., Radio F.C., Carli D., Barresi S., Brusco A., Tartaglia M., Thomas J.M., Umana L., Weiss M.M., Gotway G., Stuurman K.E., Thompson M.L., McWalter K., Stumpel C.T., Stevens S.J., Stegmann A.P., Tveten K., Vøllo A., Prescott T., Fagerberg C., Laulund L.W., Larsen M.J., Byler M., Lebel R.R., Hurst A.C., Dean J., Schrier Vergano S.A., Norman J., Mercimek-Andrews S., Neira J., Van Allen M.I., Longo N., Sellars E., Louie R.J., Cathey S.S., Brokamp E., Heron D., Snyder M., Vanderver A., Simon C., de la Cruz X., Padilla N., Crump J.G., Chung W., Garcia B., Hakonarson H.H, & Bhoj E.J. (2020). Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients. Science Advances, 6(49), eabc9207.

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Western Blot Analysis of DIMT1 Protein

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Protein collection and western blot analysis were performed as previously described (25 (link)). Cells and tissue samples were lysed on ice for 30 min using RIPA lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate; Beijing Solarbio Science & Technology Co., Ltd.] before centrifuging at 4°C, 17,000 × g for 30 min. Proteins were quantified using the bicinchoninic acid assay method (Thermo Fisher Scientific, Inc.), and equivalent protein (25 µg) was loaded to each lane and separated on 10% gels using SDS-PAGE. Following this, proteins were transferred to polyvinylidene fluoride membrane and blocked with 5% skimmed milk solution at room temperature for 1 h. Subsequently, membranes were incubated with mouse anti-human primary antibodies against DIMT1 (cat. no. ab69434; Abcam) and rabbit anti-human GAPDH (cat. no. ab97627; Abcam) at 4°C overnight. After this, membranes were incubated with the rat anti-mouse (cat. no. ab6728; Abcam) or mouse anti-rabbit (cat. no. ab99697; Abcam) secondary antibody at room temperature for 1 h. Finally, protein bands were visualized using a chemiluminescence kit (cat. no. AR21PN003; AccuRef Scientific) and qualified using ImageJ software (version 1.49; National Institutes of Health).

Zhou H.Z., Chen B., Li X.J., Du J.J., Zhang N., Shao Y.X., Zhang K, & Tong Z.C. (2021). MicroRNA-545-5p regulates apoptosis, migration and invasion of osteosarcoma by targeting dimethyladenosine transferase 1. Oncology Letters, 22(5), 763.

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Immunoblotting Analysis of Autophagy Markers

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Protein was extracted from cells using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, protein was quantified using the Pierce Protein Assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol Proteins (50 µg) were separated by 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes and probed with primary antibodies: Rabbit anti-STMN1 antibody (1:100; ab52630; Abcam, Cambridge, MA, USA), rabbit anti-LC3B antibody (1:50; ab48394; Abcam), rabbit anti-Beclin1 antibody (1:100; ab62557; Abcam), rabbit anti-mammalian target of rapamycin (mTOR) antibody (1:100; ab2732; Abcam), rabbit anti-phosphorylated (p)-mTOR (1:100; ab109268; Abcam) or rabbit anti-GAPDH antibody (1:50; ab9485; Abcam) at 4°C overnight. Membranes were subsequently incubated with mouse anti-rabbit secondary antibody (1:10,000; ab99697; Abcam) at room temperature for 40 min. The protein bands were visualized by the Amersham enhanced chemiluminescence system (RPN998; GE Healthcare Life Sciences, Chalfont, UK). Data was analyzed by densitometry using Image-Pro plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and were normalized to GAPDH expression.

Wang Z., He R., Xia H., Wei Y, & Wu S. (2017). Knockdown of STMN1 enhances osteosarcoma cell chemosensitivity through inhibition of autophagy. Oncology Letters, 13(5), 3465-3470.

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RXRα Immunoprecipitation from Pre-Adipocytes

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Brown pre-adipocytes were lysed according to the immunoblotting procedure, and samples were diluted to equal protein concentrations. Samples were then incubated with RXRα (D6H10) antibody (#3085, Cell Signalling Technology) or normal rabbit IgG (#2729, Cell Signalling Technology) overnight at 4°C while rotating. Immunocomplexes were captured by incubation with Protein A Sepharose beads for another 4 h. The beads were washed 3 times in TBST and eluted using Laemmli buffer. For tissue samples, the beads were washed in lysis buffer instead. Immunoblotting was performed using a secondary antibody (ab99697, Abcam) specific to the light chain of rabbit IgG to avoid co-detection of IgG heavy chain from the primary antibody.

Ardenkjær-Larsen J., Rupar K., Sinkevičiūtė G., Petersen P.S., Villarroel J., Lundh M., Barrès R., Rabiee A, & Emanuelli B. (2020). Insulin-induced serine 22 phosphorylation of retinoid X receptor alpha is dispensable for adipogenesis in brown adipocytes. Adipocyte, 9(1), 142-152.

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