Ab94396

Manufactured by Abcam

Sourced in United Kingdom

Ab94396 is a lab equipment product. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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14 protocols using ab94396

Quantitative Protein Analysis Protocol

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The total protein content was extracted using the RIPA lysis buffer (Beyotime) with protease inhibitors, followed by protein concentration quantitation using a BCA kit (20201ES76, Yeason, Shanghai, China). Protein was separated by PAGE and then transferred onto PVDF membrane by a wet transfer. Following blocking with 5% BSA, membranes were probed with the primary antibodies against SOX5 (ab94396, Abcam, 1:1000), EZH2 (ab186006, Abcam, 1:2000), osteocalcin (OCN; ab133612, Abcam, 1:1000), Runx2 (ab236639, Abcam, 1:1000), Collagen I (ab34710, Abcam, 1:2000), GAPDH (ab8245, Abcam, 1:3000; internal reference) overnight at 4 °C. The membrane was then re-probed with diluted secondary antibodies against HRP-labeled goat anti-rabbit IgG (ab6721, Abcam) or goat anti-mouse IgG (ab6789, Abcam) for 1 h at room temperature. Following visualization in chemiluminescence reagent, protein quantitative analysis was conducted by the ImageJ software.

Fang S., Liu Z., Wu S., Chen X., You M., Li Y., Yang F., Zhang S., Lai Y., Liu P., Jiang W, & Chen P. (2022). Pro-angiognetic and pro-osteogenic effects of human umbilical cord mesenchymal stem cell-derived exosomal miR-21-5p in osteonecrosis of the femoral head. Cell Death Discovery, 8, 226.

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ChIP Assay for SOX5 Enrichment

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ChIP assay was conducted using the EZ-Magna ChIP TMA kit (Millipore, Billerica, MA) as described previously [30 (link)]. HEK293T cells were cross-linked with 1% formaldehyde. Cells were supplemented with the protease inhibitor and ultrasonicated to obtain 200–1000 bp chromatin fragments. DNA fragments were added into 900 μL of ChIP Dilution Buffer and 20 μL of 50× PIC. After centrifugation, the supernatant was collected as Input. DNA fragments were immunoprecipitated with 1 μL of rabbit anti-SOX5 (ab94396, Abcam), while the NC group was supplemented with 1 μL of rabbit anti-IgG (ab172730, Abcam). Next, 20 μL of 5 M NaCl was added for de-crosslinking to recover the DNA content, from which the enriched chromatin fragments were detected by fluorescence real-time PCR (F: 5′-GCTAGTTATTAAATTCAT-3′, R: 5′-GTCATGTAAACTGAGAT-3′).

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Immunostaining of Chondrocyte Transcription Factors

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Primary cell cultures were fixed for 15 min at room temperature with 3% formaldehyde/PBS. The fixed cultures were washed and permeabilized with 0.1% Triton X‐100/PBS at room temperature for 10 min, incubated with primary antibodies diluted in PBS at room temperature for 1 hr, washed with PBS, and then incubated with regular secondary antibodies conjugated to Alexa 405 (Life Technologies) for 1 hr at room temperature. The following primary and secondary antibodies were used: Anti‐SOX9 antibody (Merck Millipore, AB5535), Anti‐SOX5 antibody (Abcam, ab94396), Anti‐SOX6 antibody (Abcam, ab30455), Anti‐p21 antibody (Cell Signaling Technology, 2946), Goat anti‐Rabbit IgG H + L Alexa Fluor 405 (Thermo Fisher, A‐31556), Goat anti‐mouse IgG H + L Alexa Fluor 405 (Abcam, ab175660).

Kitajima K., Kawahira N., Lee S., Tamura K., Morishita Y, & Ohtsuka D. (2021). Light‐induced local gene expression in primary chick cell culture system. Development, Growth & Differentiation, 63(3), 189-198.

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Embryonic Cartilage Characterization

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All embryos were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. The embryos were processed and sectioned into 7-μm thick paraffin sections as described previously (36 (link)). Hematoxylin and eosin staining and immunofluorescence staining of paraffin sections were performed as previously described. Primary antibodies used for immunofluorescence staining were as follows: Sox9 (1:250, Abcam; ab185966), Sox5 (1:200, Abcam; ab94396), Sox6 (1:200, Abcam; ab30455), Runx2 (1:200, Santa Cruz; sc-390351), Col2a1 (1:500, Invitrogen; MA5-12789), and GFP (1:500, Abcam; ab6673). Slides were visualized by using a LAS X imaging System (Leica).

Zhao X., Tang L., Le T.P., Nguyen B.H., Chen W., Zheng M., Yamaguchi H., Dawson B., You S., Martinez-Traverso I.M., Erhardt S., Wang J., Li M., Martin J.F., Lee B.H., Komatsu Y, & Wang J. (2022). Yap and Taz promote osteogenesis and prevent chondrogenesis in neural crest cells in vitro and in vivo. Science signaling, 15(757), eabn9009.

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Chromatin Immunoprecipitation of Sox5 in Chick Neural Tube

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Trunk neural tubes of stage HH14-16 embryos were dissected in ice cold PBS and homogenized in isotonic buffer (10mM Tris pH 7.5, 3mM CaCl₂, 0.25M Sucrose, 0.5% Triton, 1mM DTT, supplemented with Protease inhibitor cocktail, PMSF and Sodium butyrate). Chromatin was subsequently cross-linked by addition of formaldehyde to a final concentration of 1%. Cross-linking was stopped by addition of 125mM glycine. The chromatin isolation procedure was performed as previously described (Hauser et al., 2002 (link)). For ChIP, equal amounts of sonicated chromatin were diluted 10-fold and precipitated overnight with the following antibodies: anti Sox5 (Abcam ab94396, rabbit, ChIP grade), anti rabbit control IgG (Abcam ab46548, ChIP grade). Chromatin–antibody complexes were isolated with protein A magnetic beads (Dynabeads, Invitrogen). Precipitated DNA was analyzed by real time PCR using SYBR Green (Bio-Rad) on an ABI7000 qPCR machine. Diluted Inputs were used as Standards.
The following primers were used: 10E1_M4_S1 5′-CGCTGGTAACAGAGGGGTTA-3′; 10E1_M4_AS1 5′-GGGGTCGTTTTCTTGTCCTT-3′; 10E1_M4_S2 5′-GCATCCTTCCCTATCCCTTT-3′; 10E1_M4_AS2 5′-AAATGGTGCCTTTGTGCAAT-3′; 10E1_M4_S3 5′-TGGTGTGGGTGAACAGAAGA-3′; 10E1_M4_AS3 5′-TTCTACTTGTGGGGGCACTC-3′; 10E1_M4_S4 5′-CAGGGAACAAAGAAGCCATT-3′; 10E1_M4_AS4 5′-AATCACGTTGGTGTGGTGAA-3′; Control_S 5′-GGTTGGATTTCCAGTCTCCA-3′; Control_AS 5′-TGTTTTGCTGGACAATCTGC -3′.

Murko C, & Bronner M.E. (2016). Tissue specific regulation of the chick Sox10E1 enhancer by different Sox family members. Developmental biology, 422(1), 47-57.

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Chromatin Immunoprecipitation Assay for Smad3 and Sox5

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LNCaP cells were treated with 10 ng ml⁻¹ TGF-β1for 24 h to perform Smad3 (Abcam, ab28379) CHIP assay using EpiQuik Chromatin Immunoprecipitation Kit (Epigentek, Farmingdale, NY, USA) according to the protocol. CW22RV1 cells were used to perform Sox5 (Abcam, ab94396) CHIP assay. PCR was performed using primers specific for the Smad3 or Sox5 binding region in the Sox5 or Twist1 promoter. Sox5 promoter-Forward: 5′-AGTATGGGAGACGTGTTAAATGAGT-3′.
Sox5 promoter-Reverse: 5′-ACTTCCAGCAGCGGAGTCTG-3′. Twist1 promoter-Forward: 5′-CTTAGGCGCTATCAAATTCCC-3′, Twist1 promoter-Reverse: 5′-AGCGACAGCAGCAATGGCAAC-3′.

Hu J., Tian J., Zhu S., Sun L., Yu J., Tian H., Dong Q., Luo Q., Jiang N., Niu Y, & Shang Z. (2017). Sox5 contributes to prostate cancer metastasis and is a master regulator of TGF-β-induced epithelial mesenchymal transition through controlling Twist1 expression. British Journal of Cancer, 118(1), 88-97.

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Retrograde Labeling of Subcerebral Projection Neurons

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For retrograde labeling, anesthetized P0 pups were injected with Alexa Fluor 555-conjugated CTB (1 mg/ml; Invitrogen, volume injected: 300 µl) under ultrasound guidance using a Vevo 770 ultrasound backscatter microscopy system (Visual Sonics). Subcerebral injections were performed at the midbrain–hindbrain junction using a nanojector (Nanoject II Auto-Nanoliter Injector, Drummond Scientific Company 3-000-204) to label all SCPNs, including corticopontine projection neurons and corticospinal motor neurons. Injected pups were perfused at P7, and brains were collected and 40-µm-thick sections were cut at the cryostat and either directly incubated for 10 min with DAPI and mounted with ROTI Mount FluorCare (Roth) or treated for immunostaining. In the last case, slides were incubated with the blocking solution (10% goat serum and 0.3% Triton X-100) for 1 hr at RT and were then incubated with primary antibodies overnight at 4°C. The primary antibodies used were rat anti-Ctip2 (Bcl11b) (dil 1:300, Abcam ab18465), and rabbit anti-Sox5 (1:200, Abcam ab94396). Subsequent to three washes with PBS, the slides were incubated with corresponding Alexa Fluor 488 secondary antibody (1:300; Life Technologies) for 2 hr at RT. Sections were washed with PBS three times and incubated for 10 min in PBS with DAPI (1:1000) (Thermo Fisher) and mounted with ROTI Mount FluorCare (Roth).

Harb K., Richter M., Neelagandan N., Magrinelli E., Harfoush H., Kuechler K., Henis M., Hermanns-Borgmeyer I., Calderon de Anda F, & Duncan K. (2022). Pum2 and TDP-43 refine area-specific cytoarchitecture post-mitotically and modulate translation of Sox5, Bcl11b, and Rorb mRNAs in developing mouse neocortex. eLife, 11, e55199.

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Western Blot Analysis of Breast Cancer Markers

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Culture plates were washed with phosphate-buffered saline (PBS) solution and placed on ice. Total breast cancer cell proteins were extracted using mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA), containing a protease inhibitor cocktail (Thermo Fisher Scientific). Equal amounts of protein were electrophoresed using 10% sodium dodecyl sulfate-gel electrophoresis and transferred on to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were blocked using Tris-buffered saline and 0.1% Tween 20, containing 5% non-fat dry milk for 1 h, and then incubated overnight with primary antibodies. The membranes were washed three times and incubated with the secondary antibodies (Multi-sciences Biotechnology, Zhejiang, China). The membranes were then visualized using a chemiluminescence (Multi-sciences Biotechnology) detection system. The primary antibodies were used as follows: anti-Bry (Abcam, ab20680, 1:2000), anti-SOX5 (Abcam, ab94396, 1:1000), anti-E-cadherin (Abcam, ab1416, 1:50), anti-N-cadherin (Abcam, ab18203, 1:1000), snail family transcriptional repressor 1 (anti-Snai1; Abcam, ab53519, 1:1000), anti-Vimentin (Abcam, ab8978, 1:500), epithelial cell adhesion molecule (anti-EpCAM; Abcam, ab213500, 1:1000), anti-Fibronectin (Abcam, ab32419, 1:1000) and anti-β-actin (Multi-sciences Biotechnology, 70-ab008-100, 1:2000).

Chen M., Zou S., He C., Zhou J., Li S., Shen M., Cheng R., Wang D., Zou T., Yan X., Huang Y, & Shen J. (2019). Transactivation of SOX5 by Brachyury promotes breast cancer bone metastasis. Carcinogenesis, 41(5), 551-560.

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Immunofluorescence Labeling of Neuronal Markers

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Sections were washed with phosphate buffer saline (PBS) before being incubated in a blocking solution (5% donkey serum in 0.4% Triton X-100 PBS). Antibodies to NR4A2 (1:200, Abcam, ab41917 RRID:AB_776887), SATB2 (1:200, Abcam, ab51502, RRID:AB_882455), TBR1 (1:500, Abcam, ab31940, RRID: AB_2200219), NeuN (1:500, EMD Millipore, ABN78, RRID: AB_10807945), SOX5 (1:200, Abcam, ab94396, RRID: AB_10859923), CTIP2 (1:200, Abcam, ab18465, RRID: AB_2064130), Parvalbumin (1:1,000, Sigma, P3088, RRID: AB_477329) were incubated overnight at 4 °C in PBS with 2% donkey serum. After washing three times in phosphate buffer saline tween, sections were incubated with species-specific secondary antibodies for 2 h at room temperature. Secondary antibodies were obtained from Jackson ImmunoResearch. For single immunofluorescence labeling, either species-antibodies labeled with Alexa 488 or Cy3 were used. For double labeling, Alexa 488 was combined with Cy3 or Cy5. Sections were washed again three times and then mounted with Vectashield Mounting Medium for Fluorescence (Vector Laboratories Inc.).

Li H., Duque A, & Rakic P. (2023). Origin and development of the claustrum in rhesus macaque. Proceedings of the National Academy of Sciences of the United States of America, 120(28), e2220918120.

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Immunohistochemical detection of cartilage-related markers

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Sections for the detection of PAX1, PAX9, SOX5, and SOX6 were boiled in 10 mM sodium citrate at pH 6 for 10 min. Sections for the detection of ACAN were pretreated with 0.2 U/mL chondroitinase ABC (Sigma) at 37 °C for 30 min. After blocking with 3.2% skim milk/PBS, the sections were incubated with primary antibodies for 16 h, washed, and then incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 or 594 purchased from Thermo Fisher Scientific (Waltham, MA, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). The primary antibodies used were anti-ACAN (Merck Millipore, Burlington, MA, USA, AB1031; 1:500), anti-PAX1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-7744; 1:500), anti-PAX9 (Active Motif, Carlsbad, CA, USA, 61077; 1:500), anti-SOX5 (Abcam, Cambridge, UK, ab94396; 1:500), anti-SOX6 (Abcam, ab30455; 1:500), and anti-SOX9 (Merck Millipore, AB5535; 1:800). The images were captured under a Leica DMRXA microscope equipped with a Leica DC500 camera (Leica Microsystems).

Takimoto A., Kokubu C., Watanabe H., Sakuma T., Yamamoto T., Kondoh G., Hiraki Y, & Shukunami C. (2019). Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation. Scientific Reports, 9, 4605.

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