The transfected U87 cells were lysed 30 min in RIPA buffer (KeyGEN, Nanjing, China) containing protease inhibitors. A BCA kit (KeyGEN, Nanjing, China) was used to quantify the protein concentration following the manufacture’s protocol. The proteins were heated at 95 C for 5 min. Equal amounts of protein were loaded onto SDS-PAGE. After separation on the gel, the proteins were electrotransferred to a PVDF membrane. Then, 5% non-milk was used to block the membrane. And the membranes were subsequently cultured overnight with primary antibodies (dilution 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA) against phosphoinositide-3 kinase (PI3K, Cat no. 4255), phosphorylated- (p-) PI3K (Cat no. 13857), AKT (Cat no. 9272), p-AKT (Cat no. 13038), p-P70S6K (Cat no. 9204) and GAPDH (Cat no. 8884). After being washed in TBST buffer three times, the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibodies (dilution 1:300, Cat no. 7074, Cell Signaling Technology Inc., Danvers, MA, USA) for 1 h at room temperature. The membranes were washed with blocking solution and visualized using ECL detection (Thermo Fisher Scientific, Waltham, MA, USA). The signal intensity in each membrane was detected using Quantity One software and normalized relative to GAPDH.
Hrp conjugated goat anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China
The HRP-conjugated goat anti-rabbit secondary antibody is a laboratory reagent used to detect and amplify the signal of primary antibodies that were raised in rabbits. It contains horseradish peroxidase (HRP) enzyme conjugated to a goat-derived secondary antibody that binds to rabbit primary antibodies. This secondary antibody can be used in various immunoassay techniques, such as Western blotting and ELISA, to visualize and quantify the target proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Gapdh, by Proteintech (2 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Keygen Biotech (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Bca kit, by Keygen Biotech (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
0.22 mm pvdf membrane, by Merck Group (1 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Las 4000, by Cytiva (1 mentions)
Dako envision system, by Agilent Technologies (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
40 protocols using hrp conjugated goat anti rabbit secondary antibody
1
Western Blot Analysis of PI3K/AKT Pathway Activation
2
Western Blot Analysis of AGE Protein
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Total protein from cells or tissues was harvested with ice-cold RIPA buffer (10 mM Tris, 150 mM sodium chloride, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1% NP-40, pH 7.4) plus protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitor cocktail (Roche). Equivalent amounts of protein were separated by 10% or 12% SDS–PAGE and transferred onto a 0.22-mm PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were blocked with 5% skimmed milk (w/v) in TBST buffer for 1 h and incubated with the rabbit polyclonal to AGE antibody (Abcam, 1:1000) with gentle rocking overnight at 4°C. The membranes were further incubated with goat anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling Technology, 1:1500) for 1.5 h at room temperature. Finally, protein bands were imaged using an enhanced chemiluminescence detection kit (Pierce ECL Plus, Thermo Scientific) and analyzed with a luminescent image analyzer (ImageQuant LAS 4000 or Amersham Imager 600, GE Healthcare Life Sciences).
3
SATB2 Immunohistochemistry in CRC
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CRC tissues were fixed in 10% formaldehyde at 4°C for 12 h and then embedded in paraffin. Following this, tissues were cut into 6 µm sections, placed on silanized glass slides, deparaffinized in xylene and then rehydrated using ethanol gradients. The sections were then pretreated with antigen target retrieval solution [10 mmol/l citric acid monohydrate adjusted with 2 nM sodium hydroxide to (pH 6.0)] at 90°C for 40 min in citrate buffer. Endogenous peroxidase activity was blocked via incubation with methanol with 0.3% hydrogen peroxide for 30 min at room temperature. Sections were then incubated with anti-SATB2 (SATB2; 1:500; cat. no. 39229; Cell Signaling Technology, Inc.) at 4°C for 12 h. Following this, tumor tissues were incubated with goat anti-rabbit HRP-conjugated secondary antibodies (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at 37°C. Proteins were then visualized using the DAKO EnVision system (Dako; Agilent Technologies GmbH, Waldbronn, Germany) in accordance with the manufacturer's instructions and images were captured using a light microscope (magnification, ×100).
4
Quantifying MMP2 and MMP9 in TNBC
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Total protein in TNBC tissue samples and cell lines was isolated using RIPA reagent buffer (Beyotime Institute of Biotechnology) at 4°C, and the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) was used to determine protein concentration. Proteins (40 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked in 5% skimmed milk and incubated with specific primary antibodies overnight at 4°C. Primary antibodies included the anti-matrix metalloproteinase 2 (MMP2) (cat. no. 40994S; 1:1,000), anti-MMP9 (cat. no. 13667T; 1:1,000) and the loading control anti-GAPDH (cat. no. 5174T; 1:1,000), all from Cell Signaling Technology, Inc. After rinsing with TBS-0.2% Tween-20 three times, the membranes were incubated with a goat anti-rabbit HRP-conjugated secondary antibody (cat. no. 7074S; 1:3,000; Cell Signaling Technology, Inc.) at room temperature for 1.5 h. The blots were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences) and subsequently quantified using ImageJ software (version 1.52r; National Institutes of Health).
5
Western Blot Analysis of Skin Wound Proteins
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Skin wound tissues from each group on day 14 and day 21 were isolated, and samples were washed with PBS. Then, the tissue lysate was added to completely lyse the tissue, and the lysate was centrifuged at 1,200 g for 10 minutes to collect the supernatant solution containing the protein. Supernatants containing equal amounts of protein were added to a 10% SDS-PAGE gel, were electrophoresed, and were transferred to a PDVF membrane. The membrane was then blocked in 5% BSA for 2 h at room temperature and incubated overnight at 4°C in the primary antibody solution. The primary antibodies recognized GAPDH (Cell Signaling Technology, MA, USA) and EGFR (Cell Signaling Technology, MA, USA). Finally, the membrane was washed 3-4 times with TBS-T and incubated with a goat anti-rabbit HRP-conjugated secondary antibody (Cell Signaling Technology, MA, USA) for 2 h at room temperature. The intensity of protein expression was observed on an Alpha Imager scanner using chemiluminescence (Tecan, Thermo Scientific, USA), and expression analysis was performed using ImageJ software.
6
HMGB1 Protein Detection in CSF
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A total volume of 30 μl cell free CSF sample was boiled with SDS loading buffer containing protease inhibitor, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to the nitrocellulose membrane (Millipore, Darmstadt, Germany). Blots were blocked by 5 % defatted milk for 2 h, incubated with primary HMGB1 antibody (1:500, SaierBio, Tianjin, China), and further incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:2000, Cell Signaling, Beverly, MA). The membrane was visualized with an ECL Western Blotting System (Pierce Protein Research Products, Rockford, IL, USA).
7
Transcription Factor Expression Analysis
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Transfection reagent (LipofectamineTM 2000) was obtained from Invitrogen Corporation (Carlsbad, CA). Complete protease inhibitor cocktail was from Roche (Mannheim, Germany). Mouse anti-human HIF-1α monoclonal antibody was from BD Transduction Laboratories (San Diego, CA, USA). Rabbit anti-human ZEB1, Snail1, Slug, and Twist1 primary antibodies and goat anti-rabbit HRP-conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-human β-actin antibody, goat anti-mouse HRP-conjugated secondary antibody, LY294002, and lysis buffer were purchased from Beyotime Biotechnology Corporation, Shanghai (Shanghai, China). PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) and SYBR®Premix Ex TaqTM II (Tli RNaseH Plus) qPCR reagent Kit were purchased from TaKaRa Biotechnology (Dalian) Co., LTD (Dalian, China).
8
Cx43 and ERK1/2 Phosphorylation Analysis
9
PD-L1 Expression in Tumor Samples
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Protein was extracted from 15 frozen patient tumors using RIPA buffer (ThermoScientific). The tumors were collected in accordance with an IRB approved research protocol with patient/guardian informed consent as described previously62 (link). Proteins were denatured in a reducing sample buffer and run on a 12% tris gel using tris-glycine buffer. Proteins were transferred to a nitrocellulose membrane, incubated with 5% milk block, probed with antibody for PD-L1 and subsequently with either a goat anti-rabbit HRP conjugated secondary antibody (Cell Signaling) diluted in 5% bovine serum albumin. Horse radish perodixase conjugated secondary antibodies were detected using autoradiography using chemoluminescence (Amersham Enhanced Chemiluminescence Prime Western Blotting Detection Reagent). 3T3 cells and MDA-MB-231 cells were used as a negative and positive control for PD-L1 expression, respectively.
10
Western Blot Analysis of Proteins
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Cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed in cell extraction buffer (10 mM Tris HCl, pH 7.4, 1% Triton X-100, 100 mM sodium chloride, 0.1% SDS), both containing protease and phosphatase inhibitors (nacalai tesque). Protein concentrations were determined by Pierce BCA Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. Proteins (2.5–20 μg) were resolved on NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen, Grand Island, NY, USA) and then transferred onto PVDF membrane (Invitrogen). After blocking with TBS containing 0.1% Tween 20 and 1% non-fat dry skimmed milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4° C, followed by the incubation with goat anti-rabbit HRP-conjugated secondary antibody (#7074, Cell Signaling Technology) in 1:2000 dilution for 1 h at room temperature. The immune reactive bands were visualized by Amersham ECL Prime Western Blotting Detecting Reagent (GE Healthcare, Buckinghamshire, England, UK) with a ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
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