Anti bmp4

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-BMP4 is a laboratory reagent used for the detection and quantification of BMP4 (Bone Morphogenetic Protein 4) in various biological samples. It functions as a specific antibody that binds to BMP4, enabling its identification and measurement.

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6 protocols using anti bmp4

Protein Expression Analysis by Western Blot

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Cells were lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 0.1 mg/ml soybean inhibitor). Cell lysates were centrifuged at 15,000 rpm for 10 min at 4℃. Protein concentrations were measured by the BCA method using BSA as the standard. Proteins (30 µg) were separated by 8~10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% fat-free milk for 1 h in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 7.6, and 150 mM NaCl) containing 0.1% Tween 20 (TBS-T) and then incubated with the following primary rabbit or mouse antibodies: anti-BMP4, -ICAM-1, -VCAM-1 and -E-selectin (all from Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich). The antibodies were diluted 1:500~1:2,000 in 1% fat-free milk in TBS-T and incubated at 4℃ overnight. After washing, the membranes were incubated with secondary peroxidase-conjugated anti-mouse and anti-rabbit antibodies (Bio-Rad Laboratories, Hercules, CA, USA) diluted 1:1,000 in 1% fat-free milk in TBS-T at room temperature for 1 h. Detection was achieved using Pierce ECL Plus Western Blotting Substrate (ThermoFisher Scientific Inc., Rockford, IL, USA).

Hong O.K., Yoo S.J., Son J.W., Kim M.K., Baek K.H., Song K.H., Cha B.Y., Jo H, & Kwon H.S. (2016). High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 20(2), 169-175.

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Lung Protein Expression Analysis in Pups

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Lungs were dissected from pups at P5, homogenized in 1 mL 150 mM NaCl, 1% NP-40, 50 mM Tris pH 8.0 + complete Mini protease inhibitors (Roche), incubated on ice for 1 hour and spun down at 13000×g for 15 minutes at 4°C. Protein concentration was measured using a BCA kit (Thermo Scientific). Proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane. Membranes were probed with anti-BMP4 (Santa Cruz, sc-12 721, 1:1000), anti pSMAD1 (Cell Signaling, 9511 S, 1:1000) or anti-ß Actin (AbCam, ab8227, 1:10 000) primary antibodies followed by HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG2b heavy chain specific (Jackson ImmunoResearch) secondary antibodies. Immunoreactive proteins were visualized using an ECL prime kit (GE HealthCare).

McKnite A., Kim H.S., Silva J, & Christian J.L. (2023). Lack of evidence that fibrillin1 regulates bone morphogenetic protein 4 activity in kidney or lung. Developmental dynamics : an official publication of the American Association of Anatomists, 252(6), 761-769.

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Analyzing Signaling Pathways in BMP-Mediated Regulation

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Anti-p-p65, anti-p65, anti-p-ERK1/2, anti-ERK1/2, anti-p-AKT, and anti-AKT were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-GAPDH antibody was purchased from the ProteinTech Group (Chicago, IL, USA). Anti-BMP2, anti-BMP4, anti-BMPRIa, anti-BMPRII, anti-BMPRIb, and anti-occludin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant BMP2, BMP4, and noggin were purchased from Peprotech (NJ, USA). The inhibitor of NF-κB, PDTC, was purchased from Beyotime (Wuhan, China).

Chen K., Xie W., Luo B., Xiao W., Teitelbaum D.H., Yang H., Zhang K, & Zhang C. (2014). Intestinal Mucosal Barrier Is Injured by BMP2/4 via Activation of NF-κB Signals after Ischemic Reperfusion. Mediators of Inflammation, 2014, 901530.

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Tissue Morphogenesis and Signaling Pathway Analysis

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Sections were prepared as described previously [2 (link)]. Sections were stained with haematoxylin and eosin (H&E) for the tissue morphogenetic study. Immunohistochemistry analyses were performed following the manufacturer’s instructions. The primary antibodies used were anti-Wnt10b, anti-BMP4, anti-Gli1, anti-SHH, anti-SOX2, anti-β-catenin and anti-OSR2 (Santa Cruz). Images were taken using a microscope (Olympus BX43F) with an attached Olympus DP72 digital camera system.

Wang F., Li Y., Wu X., Yang M., Cong W., Fan Z., Wang J., Zhang C., Du J, & Wang S. (2017). Transcriptome analysis of coding and long non-coding RNAs highlights the regulatory network of cascade initiation of permanent molars in miniature pigs. BMC Genomics, 18, 148.

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Western Blot Analysis of BMP-4 Expression

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Correspondent volumes of CM, cells, CM + cells, or ECM elution were loaded on 12% SDS–polyacrylamide gels under reducing conditions. Samples were transferred onto a Hybond-P membrane (GE Healthcare). Anti-BMP-4 (Santa Cruz, sc-12721) or Anti-His tag (Takara, 631212) was used as primary antibodies. HRP-conjugated anti-mouse IgG (RnD Systems, HAF018) was used as secondary antibody. WesternBright Sirius HRP substrate was used for detection (Advansta, K-12043-D10). Western blots were visualized by exposing the membrane to autoradiography film.

Aykul S., Maust J, & Martinez-Hackert E. (2021). BMP-4 Extraction from Extracellular Matrix and Analysis of Heparin-Binding Properties. Molecular biotechnology, 64(2), 156-170.

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Sertoli Cell Protein Expression Analysis

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The immortalized human Sertoli cells and human primary Sertoli were lysed with RIPA buffer (Santa Cruz) for 30 min on ice. Cell lysates were cleared by centrifugation at 12,000 g for 10 min at 4°C, and the concentrations of total proteins were measured by BCA kit (Dingguo Company, China). Thirty micrograms of cell lysates from each sample were used for SDS-PAGE, and Western blots were performed according to the protocol as described previously [24 (link)]. The chosen antibody included anti-hTERT (Santa Cruz), anti-FSHR (Abcam), anti-AR (Santa Cruz), anti-GDNF, anti-SCF, anti-BMP4, anti-WT1, anti-SOX9, anti-PCNA (proliferating cell nuclear antigen, Santa Cruz), anti-3β-HSD, anti-VASA, anti-SMA, and anti-ACTB (Proteintech^TM). Replacement of primary antibodies with PBS served as negative controls (NC). After extensive washes in TBST, the images were detected by chemiluminescence (ChemiDoc^TM XRS⁺, Bio-Rad).

Wen L., Yuan Q., Sun M., Niu M., Wang H., Fu H., Zhou F., Yao C., Wang X., Li Z, & He Z. (2017). Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase. Oncotarget, 8(10), 16553-16570.

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