Protein was extracted via reagent RIPA (Takara, Dalian, China). A BCA kit was used to detect protein concentration. Then, protein extracts (30–40 µg) were separated through SDS-PAGE and electroblotted onto PVDF membrane (EMD Millipore, Billerica, MA, USA). The membrane was covered with specific antibodies (all from Abcam, Cambridge, MA, USA) at 4 °C overnight. The specific antibodies included PAX6 (1:1000, ab195045), CyclinD1 (1:200, ab16663), p21 (1:2000, ab109520), Bcl-2 (1:1000, ab182858), Cleaved caspase-3 (1:500, ab32042), and GAPDH (1:5000, ab8245). Then, the membrane was incubated with secondary antibody for 50 min at 37 °C with a dilution of 1:3000. The proteins were examined through ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA) and calculated using ImageJ software. A loading control was GAPDH.
Specific antibodies
Manufactured by Abcam
Sourced in United Kingdom, United States
Specific antibodies are immunoglobulin proteins produced by the immune system to recognize and bind to unique target molecules or antigens. These antibodies can be used in various laboratory techniques and assays to detect, quantify, or purify the target of interest.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Goat anti rabbit igg, by Abcam (2 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Takara Bio (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Ripa buffer, by Nanjing Jiancheng (1 mentions)
Western lightning ultra chemiluminescent substrate, by Bio-Rad (1 mentions)
Goat anti rabbit secondary antibody, by Abcam (1 mentions)
Pierce co immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Protein lysis buffer, by Merck Group (1 mentions)
P erk, by Abcam (1 mentions)
P stat3, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dylight conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Ecl western blot substrate, by Solarbio (1 mentions)
23 protocols using specific antibodies
1
Protein Expression Analysis via WB
2
Western Blot Analysis of BV2 Cells
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BV2 cells were harvested, washed twice with PBS, and lysed in RIPA buffer (Nanjing Jiancheng Bioengineering Institute). Protein concentrations in the lysates were quantified by the bicinchoninic acid method following the manufacturer's instructions (Nanjing KeyGen Biotech Co., Ltd.). A total of 30 µg of protein per sample was loaded and separated on 12% Mini-Protean® TGX™ gels (Bio-Rad Laboratories, Inc.) and subsequently transferred onto a polyvinylidene difluoride membrane. Prior to antibody incubations, the samples were blocked with 5% skimmed milk at room temperature for 1 h. PPM1A (dilution 1:1,000; cat. no. ab14824), JNK (dilution 1:1,000; cat. no. ab179461), phosphorylated (p)-JNK (dilution 1:3,000; cat. no. ab4821), caspase-3 (dilution 1:1,000; cat. no. ab13847), cleaved caspase-3 (dilution 1:500; cat. no. ab2302) and GAPDH (dilution 1:1,000; cat. no. ab181602) protein levels were assessed using specific antibodies (Abcam) at 4˚C overnight. Horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (dilution 1:5,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-2031) was used as the secondary antibody for 30 min at room temperature. The blot was developed using Western Lightning Ultra chemiluminescent substrate (Bio-Rad Laboratories, Inc.) in an EpiChemi3 darkroom (UVP, LLC). Image Lab 3.0 software used to analyze the results (Bio-Rad Laboratories, Inc.).
3
IHC Staining Quantification and Analysis
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IHC was conducted as previously described (24 (link)). Tissue sections (thickness, 4 μm) were deparaffinized in xylene and rehydrated through graded alcohol. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 10 min at room temperature. The specific antibodies (Abcam) were used as the primary antibodies by using a streptavidin peroxidase-conjugated method. And staining scores were observed in terms of staining intensity and percentage of positively stained cells. Staining intensity was divided into 4 grades: 0 (no staining), 1 (weak staining), 2 (intermediate staining), or 3 (strong staining). The staining percentage were divided into the following grades: 0 (<5% positive), 1 (<25% positive), 2 (25–50% positive), 3 for (51–75% positive), and 4 (>75% positive) (25 (link)). Ten independent fields (×400) were randomly selected to obtain an average score.
4
Western Blot Protein Analysis
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Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Then the membrane was blocked with 5% non-fat milk and incubated with primary antibodies at 4°C overnight. After incubation with specific antibodies (Abcam, Hong Kong, China), the blots were incubated with goat anti-rabbit secondary antibody (Abcam, Hong Kong, China) and visualized with enhanced chemiluminescence.
5
Co-Immunoprecipitation for Protein Interactions
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Co-IP was carried out using a Pierce Co-Immunoprecipitation kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, total protein was extracted from cells and quantified. A total of 5 mg of protein was incubated with 10 μg specific antibodies (Abcam) or IgG overnight at 4 °C. After elution, the recovered proteins were subjected to silver staining, mass spectrometry, and western blotting. IgG was used as a negative control.
6
STAT3 and ERK Activation Assay
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Cells were lysed in protein lysis buffer (Sigma) for protein extraction. The proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Specific antibodies against STAT, phospho-STAT3 (p-STAT3), ERK, p-ERK and actin were used (Abcam, Cambridge, UK). The signals were visualized by enhanced chemiluminescence. Recombinant human IL-6 protein (3 ng/ml) was added into the culture of SKOV3 cells with or without pre-treatment with IL-6R antibody (1500 ng/ml,).
7
Protein Interaction Analysis in Gastric Cells
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The protein lysates were extracted from cultured MKN45 and AGS cells and quantitated. The interacting proteins were co‐precipitated using the specific antibodies (Abcam), followed by SDS/PAGE. Finally, immunocomplex was analyzed with western blotting.
8
NF-κB Subunit Immunohistochemistry in Lymphoma
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Immunohistochemistry for p65 and other NF-κB subunits using specific antibodies (Abcam, Cambridge, MA) was performed on tissue microarrays of formalin-fixed, paraffin-embedded lymphoma samples using methods described previously [10 (link), 49 (link)]. The immuno-histochemical stains were assessed in 10% increments by three pathologists blinded to the clinical outcomes. Disagreements about the percentage of positive cells were resolved by joint review at a multi-headed microscope.
9
Immunostaining of Pluripotent and Cardiac Cells
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Cell cultures were fixed in 4% paraformaldehyde (Alfa Aesar, Ward Hill, MA) at 4°C for 20 minutes. Fixed cells were then permeated with 0.5% Triton X100 (Sigma-Aldrich) in PBS for 5min at room temperature. Next cells were incubated with specific antibodies (Abcam, Cambridge, UK) for pluripotency (Oct4, Nanog, Tra-1-81. Tra-1-60) and cardiac marker lineage (myosin heavy chain, connexin 43) at a 1:100 dilution, then in DyLight-conjugated secondary antibodies at a 1:500 dilution(Jackson ImmunoResearch Laboratories) and DAPI with VectaShield (Vector Laboratories, Burlingame, CA). The cell were imaged using an epifluorescence microscope (DMI 6000B, Lieca Microsystems, Wetzlar, DE).
10
Yunnanterpene G Modulates NF-κB and MAPK
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The differentiated THP-1 cells were serum-starved overnight, then pretreated with 25 μM yunnanterpene G for 4 h. Cells were lysed with buffer containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 40 mmol L−1 NaF, 10 mmol L−1 Na4P2O7, proteinase inhibitor cocktail and 1 mmol L−1 phenylmethylsulfonyl fluoride. Protein concentrations were determined by Bradford assay and equalized before loading. About 80 μg cellular proteins were separated using gradient SDS-PAGE gels and probed with specific antibodies (abcam) including NF-κB p65, phospho-NF-κB (phospho S536) p65, p38 MAPK, phospho-p38 MAPK (phospho T180/Y182), Erk1/2, phosphor-Erk1/2 (phospho Thr202/Tyr204), HRP, and GAPDH. Blots were visualized with enhanced chemiluminescence reagent.
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