Cell lysates were prepared using a NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s instructions (Pierce, USA). Samples were denatured, and equal amounts of protein were subjected to SDS-PAGE, and then transferred to nitrocellulose membrane. The membranes were blocked with 1% bovine serum albumin at room temperature for 1 h and incubated with specific indicated antibodies for 1 h, and then the membranes were incubated with the second antibodies for another 1 h. The immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system (Thermo Scientific, USA), and the band intensity was measured by densitometric analysis using Image J software.
Enhanced chemiluminescence western blotting system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The enhanced chemiluminescence Western blotting system is a lab equipment product designed for protein detection and analysis. It utilizes chemiluminescence technology to visualize and quantify protein levels in Western blot experiments.
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Lab products found in correlation
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
A5441, by Merck Group (1 mentions)
Ab195268, by Abcam (1 mentions)
Ab129085, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Lysostaphin, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti hdac8, by Cell Signaling Technology (1 mentions)
Anti gapdh, by R&D Systems (1 mentions)
Protein extraction kit, by Applygen (1 mentions)
Ab203491, by Abcam (1 mentions)
Hematoxylin, by Cell Signaling Technology (1 mentions)
Ab28486, by Abcam (1 mentions)
Ab15894, by Merck Group (1 mentions)
Polyvinylidene difluoride pvdf, by Bio-Rad (1 mentions)
Blocking serum, by Thermo Fisher Scientific (1 mentions)
8 protocols using enhanced chemiluminescence western blotting system
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Cell Proteins
3
Detection of Aap in S. epidermidis
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The presence of Aap was assessed by western blotting with a monoclonal antibody, MAB18B6, that we developed to target S. epidermidis Aap (Hu et al., 2011 (link)). The bacteria were cultivated until the OD600 reached 0.6, collected by centrifugation, and treated with 0.1 mg/ml lysostaphin (Sigma, USA). The samples were centrifuged (20,000 g) at 4°C for 30 min. The supernatants were separated using 7% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electro-transferred onto a 0.45-μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was treated with 10 ng/ml MAB18B6 and HRP-conjugated goat anti-mouse IgG (Santa Cruz, USA) as a secondary antibody, and then visualized using an enhanced chemiluminescence western blotting system (Thermo Scientific).
4
Detecting Alpha-Hemolysin Protein by Western Blot
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The collected supernatant was added 5 × loading buffer and the mixture was heated at 100°C for 5 min. Equal volumes of the mixture were separated using SDS-PAGE (10%) and transferred to polyvinylidene fluoride membrane (pore size, 0.45 μm; Millipore) by electrotransfer. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated with alpha-hemolysin polyclonal antibody (Sigma S7531) overnight at 4°C. After washing three times with PBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Santa Cruz, Santa Cruz, CA, United States). The immunoreactive bands were detected by visualization using an enhanced chemiluminescence Western blotting system (Thermo Fisher Scientific, Waltham, MA, United States).
5
Protein Expression Analysis in Nasal Mucosa
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Proteins were obtained from the nasal mucosa tissues of each mouse 1 h after the final intranasal administration of OVA or PBS. Proteins were extracted using a Protein Extraction kit (Applygen Technologies, Inc.), and total protein concentrations were measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.). A total of 20 µg protein/lane was isolated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.). The membranes were blocked with 2.5% nonfat milk at 37°C for 1 h and incubated with anti-HDAC1 (mouse; 1:1,000; cat. no. 5356), anti-H3 (rabbit; 1:1,000; cat. no. 4499), anti-H3-AcK9 (rabbit; 1:1,000; cat. no. 9649S; all from Cell Signaling Technology, Inc.), anti-HDAC8 (rabbit; 1:1,000; cat. no. 187139; Abcam) and anti-GAPDH (mouse; 1:1,000; cat. no. 686613; R&D Systems, Inc.) antibodies overnight at 4°C. Following exposure to horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies (1:200; Sigma-Aldrich; Merck KGaA) for 1 h at 37°C, immunoreactive bands were detected using an enhanced chemiluminescence western blotting system (Thermo Fisher Scientific, Inc.).
6
Comprehensive Antibody Panel for Oligodendrocyte Characterization
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Primary antibodies for β-actin (#12620), phospho-mitogenactivated protein kinase 1 and 2 (MEK1/2) (Ser217/221) (#9154), MEK1/2 (#9126), phospho-extracellular signal-regulated kinase 1 and 2 (ERK1/2) (Thr202/Tyr204) (#4370), ERK1/2 (#9102), myelin-associated glycoprotein (MAG) (#9043), myelin basic protein (MBP), #78896), and gliomaassociated oncogene homolog 1 (GLI1) (#2643) were from Cell Signaling Technology. Antibodies for 2',3'-cyclic-nucleotide 3'phosphodiesterase (CNPase) (SC-166558) and myelin oligodendrocyte glycoprotein (MOG) (SC-376138) were from Santa Cruz Biotechnology. Antibodies for oligodendrocytes transcription 2 (Olig2) (AB35128) and neuron-glial antigen 2 (NG2) (AB15894) were from Millipore. Antibodies for proteolipid protein (PLP) (ab28486) and platelet-derived growth factor receptor α (PDGFRα) (ab203491) were from Abcam Inc. The antibody for (sex determining region Y)-box 10 (Sox10) (MBS3201449) was from MyBioSource. Secondary antibodies, IHC Detection Reagents (HRP, Rabbit #8114), 3,3'-diaminobenzidine tetrahydrochloride (DAB) Substrate Kit (#8059), and hematoxylin (#14166) were obtained from Cell Signaling Technology. Polyvinylidene difluoride (PVDF), membranes, and molecular weight standards for Western blot were obtained from BIO-RAD. The enhanced chemiluminescence Western blotting system was from Thermo Fisher Scientific Inc.
7
Placental NRP-1 Protein Quantification
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The protein was extracted from placental samples and its concentration was measured by the BCA protein assay method per manufacturer's protocol (Pierce, Rockford, IL). Each sample (50 mg) was separated in 10% sodium dodecyl sulfate-polyacrylamide gel. The protein was transferred onto nitrocellulose membrane. The membrane was blocked with blocking serum (Thermoscientific, Waltham, MA) followed by incubation with mouse antihuman NRP-1 antibody (1:1000) (Abcam, Cambridge, MA) overnight at 4 C. After washing with Tris-NaCl-tween 20 buffer, the membrane was incubated with antimouse IgG (1:15000) (Abcam) at room temperature for 1 hour. Immunoreactivity was detected using an enhanced chemiluminescence Western blotting system (Thermoscientific). Qualitative analysis was performed and expressed in relation to b-actin.
8
Antibody Characterization Techniques
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Protein-A/G Sepharose (HiTrap Protein G HP, 1 ml) was purchased from GE Healthcare Life Sciences (Little Chalfont, UK). The enhanced chemiluminescence western blotting system and bicinchoninic acid protein assay kit were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Horseradish peroxidase (HRP) (H1759) and the IgG Subclass kit were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, German). 3,3',5,5'-Tetra methylbenzidine (TMB) and ELISA stop buffer were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Phosphate-buffered saline (PBS), HRP-conjugated goat anti-mouse IgG and the immunohistochemistry detection system were purchased from ZSGB-BIO (Beijing, China). PBST (0.05% Tween-20 in PBS) was used as ELISA washing buffer, Tris-buffered saline (TBS) (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) and TBST (0.05% Tween-20 in TBS) were used as western blotting washing buffer.
Cell lines. The lung cancer cell line A549, esophageal carcinoma cell line EC109 and human hepatoblastoma cell line HepG2 were maintained in our laboratory. The BCa cell line 5637 was purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). A549, EC109 and HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The 5637 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum.
Cell lines. The lung cancer cell line A549, esophageal carcinoma cell line EC109 and human hepatoblastoma cell line HepG2 were maintained in our laboratory. The BCa cell line 5637 was purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). A549, EC109 and HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The 5637 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum.
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