Cells were lysed in Staph-A buffer (1.6 mM NaH2PO4; 8.6 mM Na2HPO4; 1% Triton X-100; 0.1% SDS; 0.1% NaCl; 0.5% NaDoc; 2 mM AEBSF; 20 mg/mL each of aprotinin and leupeptin) and the obtained protein lysates (100 µg each) were subjected to SDS-PAGE electrophoresis and transferred to PVDF membrane (Millipore, Burlington, MA, USA). Blots were probed with the following monoclonal antibodies: anti-PARP (Abcam, Cambridge, MA, UK), anti-phospho-Histone γH2AX (Millipore), anti-TPX2 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-c-MYC (Santa Cruz Biotechnology); and polyclonal antibodies: anti-GOLPH3 (Abcam), anti-Beclin-1 (Novus Biologicals, Littleton, CO, USA), anti-LC3A (Cell Signaling Technology Inc., Danvers, MA, USA), anti-MYO18A (Abnova, Taipei, Taiwan), anti-Histone H2AX (Sigma, St Louis, MO, USA), and anti-MYCN (Novus Biologicals). Proteins were visualized by West Dura Extended chemiluminescent detection (Thermo Scientific, Carlsbad, CA, USA) using HRP-conjugated secondary antibodies (Thermo Scientific). Blots were re-probed with anti-β-actin (Santa Cruz Biotechnology) as loading control. Bands signal intensity was measured by densitometry using Image Lab 6.0 software (ChemiDoc, Bio-Rad, Hercules, CA, USA), and normalized to the loading control.
Anti golph3
Manufactured by Abcam
Sourced in United States
Anti-GOLPH3 is a laboratory reagent used for the detection and analysis of GOLPH3 protein. GOLPH3 is a Golgi-associated protein involved in the regulation of Golgi structure and function. This product can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of GOLPH3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Anti phospho histone γh2ax, by Merck Group (2 mentions)
Anti tpx2, by Santa Cruz Biotechnology (2 mentions)
Anti beclin 1, by Novus Biologicals (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
West dura extended chemiluminescent detection, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Anti parp, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti lc3a, by Cell Signaling Technology (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Apotome system, by Zeiss (1 mentions)
Anti cox2, by Proteintech (1 mentions)
Anti β actin, by Abcam (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Anti inos, by Proteintech (1 mentions)
Bca protein analysis kit, by Beyotime (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
6 protocols using anti golph3
1
Protein Expression Analysis by Western Blotting
2
Immunofluorescence Analysis of Neuroblastoma
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Immunofluorescence analysis was performed on formalin-fixed, paraffin-embedded NB specimens (4 μm-thick) or on cytospins as previously described [90 (link)]. The following antibodies were used: monoclonal anti-phospho-Histone γH2AX (1:20, Millipore) and anti-TPX2 (1:20, Santa Cruz Biotechnology); polyclonal anti-GOLPH3 (1:10, Abcam), and anti-Beclin-1 (1:30 Novus Biologicals). We used isotype matched non-binding mAbs in all antibody staining experiments to avoid nonspecific reactivity. Results were photographically documented using fluorescence microscope Axio Imager M2 equipped with ApoTome System (Carl Zeiss, Oberkoche, Germany). For cytospins, values represent percentages from at least 1000 counted positive and negative cells. For NB specimens, each tumor area tested contained malignant cells as assessed by histologic examination. Quantification of immunofluorescence positive tumor cells was performed on serial tissue sections, thus allowing quantification in tumor areas selected by the pathologist. Tumor cells were identified in each sample using the NB specific marker NCAM (NB56) [91 (link)]. The proportion of immunofluorescence positive cells counted was at least 100 to 1000 cells and reported in the percentage for the subsequent statistical analysis.
3
Western Blot Protein Expression Analysis
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The whole cell protein was extracted with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China, Cat#S711-001S), and the protein concentration was measured with BCA protein analysis kit (Beyotime Biotechnology, Cat#P0012). Equal amounts of total protein were loaded on 6%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the separated proteins were transferred to PVDF membrane (Merck Millipore, Darmstadt, Germany, Cat#IPVH00010). After blocking with 5% skim milk, membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with the IRDye conjugated secondary antibody for 1 h at room temperature. The antibodies used in this study were as follows: Anti-β-Actin (1:1000, Abcam, Cambridge, MA, USA, Cat#ab97626), Anti-GOLPH3 (1:1000, Abcam, Cat#ab91492), Anti-COX-2 (1:1000, Proteintech, WuHan, China, Cat#27308-1-AP), Anti-iNOS (1:1000, Proteintech, Cat#18985-1-AP) and IRDye-conjugated (680RD Goat anti-Mouse/800CW Goat anti-Rabbit) IgG secondary antibodies (1:10000, Li-COR Biosciences, Lincoln, NE, USA, Cat#926-68070/Cat#926-32211). Membranes were imaged with an Odyssey CLX Infrared Imaging System (Li-COR Biosciences).
4
GOLPH3, STIP1, c-Myc, and Cyclin D1 Expression in PDAC
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PANC1 and BXPC3 cells and six pairs of PDAC tumor specimens and adjacent non-cancerous specimens were obtained and lysed in protein lysis buffer, comprising 50 mM Tris (pH, 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mM sodium orthovanadate, 10 μM protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride. Protein were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% bovine serum albumin (BSA) in 1 × TBST for 1 h, followed by incubation with rabbit polyclonal anti-GOLPH3 (Abcam, ab236296), anti-STIP1 (Cell signaling Technology, Danvers, MA, USA; #2080), anti-c-Myc (Cell signaling Technology, #5605), anti-cyclin D1 (Cell signaling Technology, #2978), or mouse monoclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Cell signaling Technology, #5174) at 4 °C overnight. After washing the membranes were incubated with horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (Cell signaling Technology, #14708, 1:10000) for 1 h and then the immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL) kit (Millipore, Bedford, MA, USA). GAPDH was used as an internal control.
5
GFP Protein Interaction Profiling
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Green fluorescent protein (GFP)/VPS35/STIP1 ORF sequences were cloned into the CMV promoter-driven Flag tagged pBabe-based vector for transient expression. The Flag-tagged construct was transiently transfected into HEK293T cells. At 48 h after transfection, the cell pellet was harvested and lysed in NETN buffer (1 M Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40, 1mM DTT and proteinase inhibitor cocktail (Sigma, St. Louis, MO, USA). The proteins in the cell lysate were immunoprecipitated using anti-Flag affinity agarose beads. The agarose beads were washed in NETN buffer four times. For western blotting, cell lysate or the IP-elution was boiled with western blotting loading buffer for 10 min before SDS-PAGE and antibody probing. The antibodies used were rabbit polyclonal anti-GOLPH3 (Abcam, Cambridge, MA, USA; ab236296), rabbit polyclonal anti-flag (Sigma, F7425), and anti-flag M2 Affinity Gel (Sigma, A2220).
6
Protein Extraction and Western Blot Analysis
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Cytoplasmic and nuclear extracts were acquired using the NE-PER ® nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL, USA). Whole-cell protein was extracted with RIPA lysis buffer (Beyotime Biotechnology) and the protein concentration was determined with a BCA protein assay kit (Beyotime Biotechnology). Briefly, proteins were separated by 6-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany).
Membranes were blocked in 5% skim milk (BD/Difco, Sparks, MD, USA) for 1 h, and then incubated with different primary antibodies at 4°C overnight. The primary antibodies utilized were: anti-GOLPH3 (1:1000; Abcam), anti-EGFR (1:1000; Protein Tech Group), anti-gamma H2AX (phospho S139) (1:1000; Abcam), anti-ubiquitin For co-immunoprecipitation (co-IP), 2 g of anti-DNA-PK, anti-EGFR or control IgG antibody was incubated with 4 mg of cell lysate, followed by capturing with protein-A/G agarose. The beads were then washed extensively and suspended in SDS loading buffer for western blot analysis.
Membranes were blocked in 5% skim milk (BD/Difco, Sparks, MD, USA) for 1 h, and then incubated with different primary antibodies at 4°C overnight. The primary antibodies utilized were: anti-GOLPH3 (1:1000; Abcam), anti-EGFR (1:1000; Protein Tech Group), anti-gamma H2AX (phospho S139) (1:1000; Abcam), anti-ubiquitin For co-immunoprecipitation (co-IP), 2 g of anti-DNA-PK, anti-EGFR or control IgG antibody was incubated with 4 mg of cell lysate, followed by capturing with protein-A/G agarose. The beads were then washed extensively and suspended in SDS loading buffer for western blot analysis.
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