Brain tissue was homogenized on ice, and ice-cold RIPA lysis buffer (Beyotime) containing 1 mM PMSF (Beyotime) was added to each sample. After centrifugation at 14,000×g at 4 °C for 10 min, the supernatants were collected. The protein concentration was measured with a BCA kit (Beyotime). Approximately 50 μg of protein was separated via SDS-PAGE with an appropriate concentration of SDS. The protein was then transferred to nitrocellulose membranes. The membranes were incubated with the following primary antibodies at 4 °C overnight: mouse anti-claudin-5 (1:500, Invitrogen), mouse anti-Bax (1:1000, Cell Signaling Technology), rabbit anti-Bcl-2 (1:1000, Abcam), rabbit anti-MMP-9 (1:1000, Abcam) and mouse anti-β-actin (1:1000, Transgen). Then, the membranes were incubated with the appropriate secondary antibodies at 25 °C for 2 h: goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP antibodies (1:5000, Cell Signaling Technology). After detection with a ChemiDocTM MP imaging system (Bio-Rad), the results were analyzed with Image J software version version 1.48.
Goat anti mouse igg hrp
Manufactured by Cell Signaling Technology
Sourced in United States, Japan
Goat anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse immunoglobulin G (IgG) in immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Cell Signaling Technology (18 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Goat anti rabbit igg horseradish peroxidase hrp, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Supersignal west dura, by Thermo Fisher Scientific (2 mentions)
Anti β actin monoclonal antibody, by Merck Group (2 mentions)
Anti mouse αsyn monoclonal antibody, by Novus Biologicals (2 mentions)
Mouse anti rhoa igg, by Merck Group (2 mentions)
Western lightning ecl 2 kit, by PerkinElmer (2 mentions)
Papain, by Merck Group (2 mentions)
Recombinant human epidermal growth factor, by R&D Systems (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Human neurocult ns a proliferation supplements, by STEMCELL (2 mentions)
Neurocult ns a basal medium human, by STEMCELL (2 mentions)
Donkey anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (2 mentions)
42 protocols using goat anti mouse igg hrp
1
Western Blot Analysis of Brain Tissue
2
Extraction and Detection of α-Synuclein
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Brain tissues (1:10, w/v) were homogenized in lysis buffer (10 mM Tris-Cl, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, pH 8.0) containing Protease Inhibition Cocktail III. Soluble and insoluble αSyn proteins were subsequently lysed with two different detergents. The soluble part was first extracted with 1% Triton X-100 in the lysis buffer. The insoluble part in the remaining pellet was further extracted with lysis buffer containing 1% Triton X-100 /2% SDS. Protein concentration was determined by BCA Protein Assay kit (Thermo Fisher Scientific). Pre-casted 4–12% gradient gel (Invitrogen) was used for protein separation. The proteins on the gels were transferred onto 0.45μm PVDF membranes. The membrane was blocked in 5% milk in PBS for 20 min at room temperature. Anti-mouse αSyn monoclonal antibody (1:500 in 3% milk/PBS, Novus) was incubated overnight at 4°C followed by goat-anti-mouse IgG-HRP (1:1500 in 1% milk/PBS, Cell signaling) incubation for 1 hr at room temperature. An ECL detection system (SuperSignal™ West Dura, Thermo Fisher Scientific) was applied for signal detection. Anti β-actin monoclonal antibody (1:5000, Sigma) was used as a loading control.
3
Whole Cell Protein Extraction and Western Blotting
4
Western Blot Analysis of Protein Expression
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Cells were digested with RIPA cell lysis buffer (Beyotime Biotechnology, Shanghai, China) with protease and phosphatase inhibitor cocktails (MCE, Shanghai, China) and were collected using a cell scraper. Total protein was extracted and quantified using the bicinchoninic acid method (Thermo Fisher Scientific). Proteins (20–30 μg) were resolved using 10% PAGE (Bio-Rad, Hercules, CA, USA), transferred to a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA) at 25 V for 30 min, and blocked for 1 h in 10% nonfat milk in 1× TBS/0.1% (v/v) Tween 20 at room temperature. Primary antibodies (GAPDH, 1:1,000, #5174; Cell Signaling Technology; β-actin, 1:1,000, #4970; Cell Signaling Technology; P1-HNF4A, 1:1,000, ab41898; Abcam; P2-HNF4A, 1:1,000, PP-H6939-00; R & D Systems; and CCL15, 1:1,000, ab219388; Abcam) were added and incubated overnight at 4 °C. Secondary antibodies (goat anti-rabbit IgG, HRP #7074 or goat anti-mouse IgG, HRP, 1:2,000, #7076; Cell Signaling Technology) were incubated at room temperature for 1 h. Signals were detected using Western Lumax Light Sirius HRP substrate reagent (ZETA-Life, San Francisco, CA, USA). All data were normalized to human β-actin or GAPDH. The bands were scanned using a ChemiDocXRS + Imaging System (Bio-Rad).
5
Neurological Signaling Pathway Profiling
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Cadmium chloride, quercetin, protease inhibitor and antibody for anti-mouse TH were purchased from Sigma Aldrich, India. The antibodies for anti-rabbit Dopamine transporter (DAT), anti-rabbit vesicular monoamine transporter (VMAT-2), anti-rabbit protein kinase A (PKA), anti-rabbit DARPP-32, anti-rabbit PP1α, anti-rabbit CREB, anti-rabbit Akt, and anti-rabbit β- actin were purchased from Cell Signaling Technology, USA. Secondary antibodies for goat anti-rabbit IgG HRP, goat anti-mouse IgG-HRP were also purchased from Cell Signaling Technology, USA. Other chemicals used in the study were of analytical grade and procured from local commercial sources.
6
Detecting RhoA Activation by CNF Y
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Cells were seeded in 10 cm cell culture dishes at the concentration of 2.2 × 106 cells/dish and allowed to attach overnight. The next day, cells were washed and incubated for 4 h with 20 µg/ml of cleared lysates from Y. pseudotuberculosis expressing different CNFY derivatives at 37°C, 5% CO2. Cells were washed with cold PBS and lysed in 150 μl lysis buffer containing 50 mM Tris‐HCl (pH 7.4), 100 mM NaCl, 2 mM MgCl2, 10% NP‐40, and 0.5 mM phenyl–methyl–sulfonyl fluoride (PMSF). Cells were then scraped off and centrifuged for 30 min (11,500 g, 4°C). Sodium dodecyl sulfate (SDS) sample buffer was added to the cleared lysates and samples were separated on 12% SDS‐gel. After blotting onto a PVDF membrane, RhoA was detected using mouse anti‐RhoA IgG (Millipore) (1:1,000) as a primary antibody and followed by secondary antibody goat anti‐mouse IgG‐HRP (Cell signaling). Membranes were visualized using the Western Lightning ECL II Kit (Perkin Elmer) and exposed on X‐Ray film.
7
Quantifying PON1 Expression in HEK293 Cells
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An amount of 30 μg of total protein from the lysates of the transfected HEK293 cells was electrophoresed on 12% SDS-PAGE gels and subsequently transferred onto nitrocellulose membranes for immunoblotting. PON1 was detected using the mouse anti-DYKDDDDK Tag monoclonal antibody 9A3 (1:500, Cell Signaling Technology, Danvers, MA, USA) and a goat anti-mouse IgG-HRP (1:1000, Cell Signaling Technology). In addition, α-tubulin was detected by a rabbit anti-α-tubulin polyclonal antibody (1:1000, Cell Signaling Technology) and a goat anti-rabbit IgG-HRP (1:2000, Merck, Darmstadt, Germany). Quantification of protein levels was conducted by densitometry analysis of the immunoreactive bands using the ImageJ software (version 1.54d) [31 ].
8
IgG Glycan Analysis by Lectin Blotting
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Purified IgG (5 μg) was incubated in digestion buffer (10 mM EDTA, 10 mM cysteine in PBS (pH 7.4)) with 0.25 μg of papain (Sigma-Aldrich) for 2 h at 37 °C. Digested IgGs were separated by 10% SDS–PAGE (IgGs, 1.25 μg per lane for lectin blotting; 100 ng per lane for western blotting), and transferred onto polyvinylidene difluoride membranes by semi-dry electrophoresis. For western or lectin blotting, the membranes were incubated with either goat anti-mouse IgG-HRP (Cell Signaling Technology Japan, K.K., Tokyo, Japan, #7076) or biotin-conjugated lectin (Vector Laboratories, Inc., Burlingame, CA). Terminal α2,6 sialic acid (Sia), β1,4 galactose (Gal), N-acetylglucosamine (GlcNAc), and α-linked mannose (αMan) were detected by SNA (B-1305), ECL (B-1145), GSL II (B-1215) and ConA (B-1005S) lectin, respectively.
9
Signaling Pathway Inhibition Assay
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RPMI-1640 medium, fetal bovine serum (FBS), and antibiotics were purchased from GIBCO-BRL (Grand Island, NY, USA). The antibodies against phospho-Akt (phospho-Ser473, pAkt), total ERK (tERK), phospho-ERK (p-ERK), total tJNK, phospho-JNK (p-JNK), NF-κB p65, iNOS, goat anti-mouse IgG-HRP, goat anti- rabbit IgG-HRP, and donkey anti-goat IgG-HRP, were obtained from Cell Signaling Technology (Danvers, MA, USA). The Lamin B and total Akt (tAkt) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The general signaling inhibitors, LY294002 (PI3K inhibitor), PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor), were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).
10
Culturing Patient-Derived Glioblastoma Stem Cells
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The patient-derived GSCs were well characterized (Table 1 ) and cultured in NS-A medium (90% NeuroCult NS-A Basal Medium Human plus 10% Human NeuroCult NS-A proliferation Supplements, StemCell Technologies, Vancouver, British Columbia, Canada in 3D sphere as we described before [45 (link)]. Complete medium was supplied with recombinant human epidermal growth factor (R&D System, Minneapolis, MN, USA) and 100 units/mL penicillin-100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). Anti-TUSC3 and anti-MGMT antibodies were obtained from Abcam (Cambridge, United Kingdom); anti-β-actin IgG-HRP was obtained from Santa Cruz Biotech (Dallas, TX, USA) and anti-DNMT1, Goat anti-Rabbit IgG-HRP and Goat anti-mouse IgG-HRP were obtained from CellSignaling Technology (Danvers, MA, USA). Temozolomide (TMZ) was obtained from Sigma (St. Louis, MO, USA). 5-Azacitidine (5-Aza) and Lomeguatrib were obtained from Selleckchem (Houston, TX, USA).
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