Donkey anti goat igg hrp

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom, Germany

Donkey anti-goat IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to goat primary antibodies in immunoassays and immunohistochemistry applications.

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Lab products found in correlation

97 protocols using donkey anti goat igg hrp

Investigating GFI1 Transcription Factor Regulation

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Primary human CD34⁺ cells, AML cell lines transduced with eGFP and GFI1-eGFP lentiviral vectors were sorted and nuclear proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Darmstadt, Germany). Antibodies used for Western blotting included goat polyclonal antibody against GFI1 (N-20) (1:1000, G6670, Sigma-Aldrich, Darmstadt, Germany), rabbit polyclonal antibody against PCNA (1:500, sc-7907, Santa Cruz Biotechnologies, Minneapolis, USA), donkey anti-goat IgG-HRP (1:5000, sc-2020, Santa Cruz Biotechnologies) and Goat anti-rabbit IgG-HRP (1:2500, sc-2030, Santa Cruz Biotechnologies). ECL Western blot analysis system (Amersham) was used for detection.

Hönes J.M., Thivakaran A., Botezatu L., Patnana P., Castro S.V., Al-Matary Y.S., Schütte J., Fischer K.B., Vassen L., Görgens A., Dührsen U., Giebel B, & Khandanpour C. (2017). Enforced GFI1 expression impedes human and murine leukemic cell growth. Scientific Reports, 7, 15720.

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TREM-1 Immunoblot Analysis Protocol

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SDS-PAGE and subsequent Immunoblot was performed following standard methods. Briefly samples were mixed with sample loading buffer including β-mercaptoethanol and heated at 65°C for 15 minutes. Following electrophoresis on a Mini-Protean TGX 4-20% gradient gel, bands were blotted onto 0.45 micron nitrocellulose membrane and stained with Ponceau-S for total protein. Membrane was blocked for 4 hours at room temperature in a buffer with 3% BSA and incubated overnight at 4°C with either 0.8 μg/ml goat anti-human TREM-1 polyclonal antibody (R&D Systems #AF1278) or 2.0 ug/ml rabbit anti-human TREM-1sv polyclonal antibody. Secondary antibodies used were donkey anti-goat IgG-HRP and donkey anti-rabbit IgG-HRP respectively (both from Santa Cruz Biotechnologies) at a dilution of 1:15000. Blots were developed with Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific) and visualized on film.

Baruah S., Keck K., Vrenios M., Pope M., Pearl M., Doerschug K, & Klesney-Tait J. (2015). Identification of a novel splice variant isoform of TREM-1 in human neutrophil granules. Journal of immunology (Baltimore, Md. : 1950), 195(12), 5725-5731.

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Western Blot Analysis of Protein Levels

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Whole cell lysates were prepared using EBC₂₅₀ lysis buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, 2 µg/mL aprotinin, 40 mM NaF, and 0.5 mM NaVO₄). Equal amounts of total protein were separated by SDS-PAGE and then transferred onto PVDF membranes and hybridized to a specific primary antibody and HRP-conjugated secondary antibody for subsequent detection by ECL (Pierce). Primary antibodies and dilutions used: Rb 1:500 (G3-245, BD Bioscience); myogenin 1:400 (F5D, Santa Cruz); actin 1:1000 (C-11, Santa Cruz); murine MDM2 (mixture of 2A10, 4B2, and 4B11; 1.0 mL each in 20 mL), and MyHC 1:400 (F-20, DSHB). Secondary antibodies were purchased from Santa Cruz Biotechnology and used at 1:3000 dilutions, including goat anti-mouse IgG-HRP (sc-2005), goat anti-rabbit IgG-HRP (sc-2004), and donkey anti-goat IgG-HRP (sc-2020).

Walsh E.M., Niu M., Bergholz J, & Xiao Z.X. (2015). Nutlin-3 down-regulates Retinoblastoma protein expression and inhibits muscle cell differentiation. Biochemical and biophysical research communications, 461(2), 293-299.

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Signaling Pathway Inhibitors Protocol

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RU486 (MIF), AG1498, wortmannin, rapamycin, and pyrazolopyrimidine compound (PP1) were purchased from EMD Chemicals (Gibbstown, NJ, USA). BpV (phen) was from Thermo Fisher Scientific (Pittsburgh, PA, USA). Anti-mPRα goat polyclonal IgG, anti-MMP9 goat polyclonal IgG, anti-GAPDH goat polyclonal IgG, anti-mPRα blocking peptide, donkey anti-goat IgG-HRP, goat anti-rabbit IgG-HRP, and anti-mouse IgG were purchased from Santa Cruz Biotechnology (CA, USA). Anti-VEGF polyclonal antibody was from Abcom (Cambridge, MA, USA). Anti-KCNMA1 rabbit polyclonal antibody was from Millipore (Billerica, MA, USA). Anti-FAK rabbit polyclonal and anti-p-FAK rabbit polyclonal IgG were from Cell Signaling (Danvers, MA, USA). P4-BSA-FITC conjugate and anti-α-tubulin mouse monoclonal IgM were purchased from Sigma (St. Louis, MO, USA).

Xie M., Zhou L., Chen X., Gainey L.O., Xiao J., Nanes M.S., Hou A., You S, & Chen Q. (2015). Progesterone and Src Family Inhibitor PP1 Synergistically Inhibit Cell Migration and Invasion of Human Basal Phenotype Breast Cancer Cells. BioMed Research International, 2015, 426429.

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Antibody Characterization for Protein Analysis

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For Western blotting the following antibodies were used as follows: MxB (Santa Cruz sc-271527; RRID: AB_10649506) at 1:200, Sec62 (Abcam ab168843) at 1:2000, ZAP/ZC3HAV1 (Proteintech #16820–1-AP; RRID: AB_2728733) at 1:5000, N4BP1 (Cohesion Biosciences #CPA2415) at 1:1000, tubulin (Sigma T6199; RRID: AB_477583) at 1:1000 and actin (Sigma A2066; RRID: AB_476693) at 1:5000. Secondary antibodies were used as follows: 1:5000 donkey anti-goat IgG-HRP (Santa Cruz Biotechnology sc-2020; RRID: AB_631728) and 1:5000 goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology sc-2004; RRID: AB_631746). For flow cytometry, antibodies were used as follows: CD4 (BD Pharmingen 555349; RRID: AB_398593) 1:50, CXCR4 (eBioscience 17-9999-42; RRID: AB_1724113) 1:50, CD-169 (BioLegend 346003; RRID: AB_2189038) 1:50, TLR2 (BioLegend 309707; RRID: AB_314777) 1:100, Tetherin (BioLegend 348410; RRID: AB_2067121) 1:50.

OhAinle M., Helms L., Vermeire J., Roesch F., Humes D., Basom R., Delrow J.J., Overbaugh J, & Emerman M. (2018). A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV. eLife, 7, e39823.

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Comprehensive Protein Analysis Protocol

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Total cell lysate extraction and western blotting were performed using a previously described protocol.14, 15 The extracted protein was quantitated using a protein assay (Bio‐Rad Laboratories) and was transferred to a membrane using a transfer system (Bio‐Rad Laboratories, Hercules, CA, USA). Then, the membranes were probed with primary antibodies diluted with 5% BSA overnight at 4°C. After washing more than 3 times using PBS‐tween, the membranes were incubated with the secondary antibodies for 1 hour at 25°C. To detect specific signals, we examined the membrane using the ECL Prime Western Blotting Detection System (GE Healthcare) and LAS‐3000 (Fuji Film). The primary antibodies were as follows: p‐HER2 (Tyr1221/1222), HER2, p‐EGFR (Tyr1068), EGFR, p‐MAPK (Erk1/2) (Thr202/Tyr204), MAPK (Erk1/2), p‐AKT (Ser473), AKT, IGF‐I receptor (IGF‐1R), p‐IGF‐I receptor (phospho‐IGF‐1R) (Tyr1135/1136), MET, p‐MET(Tyr1234/1235), YES, Src, p‐Src (Tyr416), ALDH1A1, ABCB1, E‐cadherin, vimentin and p‐AXL(Tyr702) (Cell Signaling Technology, Danvers, MA, USA), AXL (R&D Systems, Minneapolis, MN, USA), and actin (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies used in this study were as follows: goat anti–mouse IgG‐HRP, goat anti‐rat IgG‐HRP and donkey anti–goat IgG‐HRP (Santa Cruz Biotechnology).

Yoshioka T., Shien K., Takeda T., Takahashi Y., Kurihara E., Ogoshi Y., Namba K., Torigoe H., Sato H., Tomida S., Yamamoto H., Soh J., Fujiwara T, & Toyooka S. (2019). Acquired resistance mechanisms to afatinib in HER2‐amplified gastric cancer cells. Cancer Science, 110(8), 2549-2557.

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Exosomal Protein Analysis via Immunoblotting

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Exosomal markers in both pooled samples and the individual fractions were determined by immunoblotting for FLOT-1 (details described subsequently). Exosomal protein concentration was quantified by using a Bicinchoninic Acid reagent kit (Sigma Aldrich, Castle Hill, NSW, Australia). 10 μg of exosome protein (singular fractions and pooled fractions 7–10) was incubated for 10 min at 70°C in reducing agent (NuPAGE Sample Reducing Agent, Life Technologies Australia Pty Ltd, Mulgrave, VIC, Australia) and loading buffer (NuPAGE LDS buffer, Life Technologies Australia Pty Ltd). Reduced proteins were electrophoresed and transferred onto a polyvinyl difluoride (PVDF; Bio-Rad Laboratories Pty Ltd, Australia) membrane as previously described [1 (link)]. Membranes were incubated for 1 hour in 2% BSA and probed overnight with primary goat polyclonal antibody anti-Flotillin 1 (FLOT-1) (ab13493 Abcam, Cambridge, UK) at 4°C, followed by secondary donkey anti-goat IgG-HRP (1 : 1000; sc-2020, Santa Cruz Biotechnology, CA, USA). SuperSignal West Dura-Extended Duration Substrate (Thermo Fisher Scientific, Australia Pty Ltd) was used for development, and the signal visualized on X-ray film (Agfa, Mortsel, Belgium) was developed using a Konica Minolta SRX-101A processor (Konica Minolta Medical and Graphic Inc, Japan) (method derived from [1 (link), 4 ]).

Vaswani K., Mitchell M.D., Holland O.J., Qin Koh Y., Hill R.J., Harb T., Davies P.S, & Peiris H. (2019). A Method for the Isolation of Exosomes from Human and Bovine Milk. Journal of Nutrition and Metabolism, 2019, 5764740.

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Antibody Inventory for EMT Analysis

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Antibodies against E-cadherin, N-cadherin, α-catenin, β-catenin and γ-catenin were purchased from BD Science Transduction Laboratories (Lexington, KY, USA). Antibodies against Desmoplakin I&II, vimentin (AB-2), Epithelial Specific Antigen (Ab-2) and Epithelial Membrane Antigen (Ab-2) were obtained from LabVision Corporation (Fremont, CA, USA). The β-actin antibody, the goat anti-mouse IgG-HRP, the goat anti-rabbit IgG-HRP and the donkey anti-goat IgG-HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-rabbit and mouse Alexa Fluor 488 antibodies were purchased from Invitrogen Life Technologies (Grand Island, NY, USA).

An J., Lv J., Li A., Qiao J., Fang L., Li Z., Li B., Zhao W., Chen H, & Wang L. (2015). Constitutive expression of Bcl-2 induces epithelial-Mesenchymal transition in mammary epithelial cells. BMC Cancer, 15, 476.

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Analysis of Cell Tight Junction Proteins

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Roswell Park Memorial Institute (RPMI)-1640 medium (LM011-01), fetal bovine serum (FBS; S001-07), and trypsin (LS015-01) were obtained from WELGENE Inc. Phosphate buffered saline (PBS; 17-517Q) was obtained from Lonza. ProLong® Gold antifade reagent (P36931) containing 4,6-diamidino-2-phenylindole (DAPI), and antibodies for claudin-1 (51-9000) and occludin (40-6100) were purchased from Invitrogen. Antibodies for phospho-extracellular signal-regulated kinases1/2 (p-ERK1/2) (9101) and ERK1/2 (9102) were purchased from Cell Signaling Technology. Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778), p65 (sc-372), Lamin B (sc-6216), goat anti-rabbit IgG-HRP (sc-2030), donkey anti-goat IgG-HRP (sc-2020), and an ERK1/2 inhibitor [PD98059 (PD); sc-3532] were obtained from Santa Cruz Biotechnology, Inc. DyLight™-488-conjugated anti-IgG (A120-10872) was purchased from Bethyl Laboratories. Anhydrous aluminum chloride (563919; Al 20.24%, Cl 79.76%) an nuclear factor-kappa B (

NF- κ B

) inhibitor [Bay11-7085 (Bay); B5681], and Pierce bicinchoninic acid (BCA) protein assay kit (23225) were purchased from Sigma-Aldrich. Nitrocellulose membrane (10600002) was obtained from (GE Healthcare Bio-Sciences).

Jeong C.H., Kwon H.C., Kim D.H., Cheng W.N., Kang S., Shin D.M., Yune J.H., Yoon J.E., Chang Y.H., Sohn H, & Han S.G. (2020). Effects of Aluminum on the Integrity of the Intestinal Epithelium: An in Vitro and in Vivo Study. Environmental Health Perspectives, 128(1), 017013.

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Western Blot Analysis of CD44 and CD146

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Protein lystaes (40 μg) were prepared as previously described (Ouhtit et al., 2007) and boiled for 5 min in an equal volume of reducing buffer (5 mmol/L of Tris/HCl (pH 7.4), 4% (w/v) sodium dodecyl sulfate, 20% (v/v) glycerol, 10% (v/v) mercaptoethanol, 0.2% (w/v) bromophenol blue), resolved on 12% polyacrylamide gels and electroblotted onto nitrocellulose membranes. Membranes were probed with mouse monoclonal anti-CD44 (1:1000 dilution; R&D Biosystems, MN), a mouse monoclonal anti-CD146 (1:500 dilution; Novocastra HD, Leica Biosystems, UK), and goat anti-actin antibodies (1:500 dilution; Santa Cruz Biotechnology, CA), followed by incubation with donkey anti-mouse (1:2000 dilution; Santa Cruz Biotechnology, CA) and donkey anti-goat IgG-HRP (1:2000 dilution; Santa Cruz Biotechnology, CA) secondary antibodies. The presence of the protein was detected using the West Femto Supersignal chemiluminescence kit (Thermo Scientific, IL).

Ouhtit A., Abdraboh M.E., Hollenbach A.D., Zayed H, & Raj M.H. (2017). CD146, a novel target of CD44-signaling, suppresses breast tumor cell invasion. Cell Communication and Signaling : CCS, 15, 45.

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