Bovine serum albumin (BSA), ovalbumin (OVA), potassium carbonate, and chloroauric acid solution were obtained from Sigma (St. Louis, MO). Protein-G columns and goat anti-rabbit IgG were supplied by Solarbio Corporation (Beijing, China). Nitrocellulose (NC) films were purchased from Sartorius (Gottingen, Germany). Polyester fiber membranes, special absorbent paper, plastic cards, and glass fiber (GF) membranes were bought from Shanghai Kinbio Tech Co., Ltd. (Shanghai, China). Serum and coating antigen were developed in a previous study [23 (link)]. Standards (Dicamba; 5-hydroxydicamba; 2,3,5-trichlorobenzoic acid; 2,3,6-trichlorobenzoic acid; clopyralid; picloram; chloramben and chlorfenac) were obtained from Thermo Fisher Scientific (Rockford, IL), Sigma-Aldrich (St. Louis, MO) or Chem Service, Inc. (West Chester, PA). The solvents and reagents used in this study were all of analytical grade.
Goat anti rabbit igg
Manufactured by Solarbio
Sourced in China
Goat anti-rabbit IgG is a secondary antibody used in immunoassays and other immunological techniques to detect the presence of rabbit primary antibodies. It binds to the Fc region of rabbit immunoglobulin G (IgG) molecules.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by Solarbio (5 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Bca kit, by Solarbio (2 mentions)
Chloroauric acid solution, by Merck Group (1 mentions)
Gapdh, by Solarbio (1 mentions)
P ampkα, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
P p44 42 mapk, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Super ecl prime, by US Everbright (1 mentions)
Ecl western blotting substrate, by Solarbio (1 mentions)
Hrp conjugated goat anti mouse, by Solarbio (1 mentions)
Gapdh mouse monoclonal antibody, by Proteintech (1 mentions)
Cox 2, by ABclonal (1 mentions)
Skim milk, by Sangon (1 mentions)
β catenin, by Elabscience (1 mentions)
11 protocols using goat anti rabbit igg
1
Immunoassay for Dicamba Detection
2
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Solarbio, Beijing, China) with Protein phosphatase inhibitor (Biomed, Beijing, China) and phenylmethylsulfonyl fluoride (PMSF, Biomed) was used to extract total protein lysate. Lysates were run on 10% SDS-PAGE gels, and protein bands were transferred to 0.45μm polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), then blocked with 5% skim milk at room temperature for 1 hour. The membrane was incubated with primary antibodies (GNA15, Novus Biologicals, Centennial, USA, 1:1000; GAPDH, Solarbio, 1:1000; p-P38 MAPK, P38 MAPK, p-CREB, CREB, p-MAPKAPK2Thr222, MAPKAPK2Thr222, p53, cleaved-PARP, cleaved-Caspase3, p27 Kip1, Cyclin D1, CDK4, p-p44/42 MAPK, p-AMPKα, p-Akt, p-Smad3, LC3A/B, Cell Signaling Technology [CST], MA, USA, 1:1000) overnight at 4°C and probed with secondary antibodies (goat anti-rabbit IgG, goat anti-mouse IgG, Solarbio, 1:1000) at room temperature for 1 hour. The immunoreactive bands were defined using Super ECL Prime (US EVERBRIGHT, Suzhou, China).
3
Protein Quantification and Western Blot Analysis
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Protein quantification was measured using BCA Protein Assay Kit (Solarbio). Equal amounts
of protein (10 µg) from each sample were separated by 8% or 15% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto
PVDF (polyvinylidene difluoride, Millipore, Bedford, MA, USA) membranes. The membranes
were blocked with 5% skim milk (Sangon Biotech, Shanghai, China) for 1 h at room
temperature and incubated with primary antibodies: cholecystokinin rabbit polyclonal
antibody (CCK, 1:1,000, NOVUS, St. Charles, MO, USA), somatostatin rabbit polyclonal
antibody (SST, 1:500, ABclonal, Wuhan, China), Cleaved Caspase-3 rabbit polyclonal
antibody (1:1,000, CST, Danvers, MA, USA), neuropeptide Y rabbit polyclonal antibody (NPY,
1:1,000, Affinity, Changzhou, China), Cleaved PARP rabbit polyclonal antibody (1:1,000,
CST), cyclooxygenase-2 rabbit polyclonal antibody (COX-2, 1:500,
ABclonal) and GAPDH mouse monoclonal antibody (internal control,
1:10,000, proteintech, Wuhan, China), overnight at 4°C. After washing, the membranes were
incubated with HRP-conjugated goat anti-mouse (1:3,000, Solarbio) or goat anti-rabbit IgG
(1:3,000, Solarbio) for 1 h at 37°C and visualized by using ECL Western Blotting Substrate
(Solarbio).
of protein (10 µg) from each sample were separated by 8% or 15% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto
PVDF (polyvinylidene difluoride, Millipore, Bedford, MA, USA) membranes. The membranes
were blocked with 5% skim milk (Sangon Biotech, Shanghai, China) for 1 h at room
temperature and incubated with primary antibodies: cholecystokinin rabbit polyclonal
antibody (CCK, 1:1,000, NOVUS, St. Charles, MO, USA), somatostatin rabbit polyclonal
antibody (SST, 1:500, ABclonal, Wuhan, China), Cleaved Caspase-3 rabbit polyclonal
antibody (1:1,000, CST, Danvers, MA, USA), neuropeptide Y rabbit polyclonal antibody (NPY,
1:1,000, Affinity, Changzhou, China), Cleaved PARP rabbit polyclonal antibody (1:1,000,
CST), cyclooxygenase-2 rabbit polyclonal antibody (COX-2, 1:500,
ABclonal) and GAPDH mouse monoclonal antibody (internal control,
1:10,000, proteintech, Wuhan, China), overnight at 4°C. After washing, the membranes were
incubated with HRP-conjugated goat anti-mouse (1:3,000, Solarbio) or goat anti-rabbit IgG
(1:3,000, Solarbio) for 1 h at 37°C and visualized by using ECL Western Blotting Substrate
(Solarbio).
4
Protein Expression Analysis by Western Blot
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Cells were lysed in RIPA buffer (Solarbio) containing PMSF. Protein quantification was performed using the BCA Protein Quantification Kit (Solarbio). The protein sample was blended with the protein loading buffer and heated at 99°C for 5 minutes. The denatured proteins were separated by 10% SDS–PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was placed in 5% skim milk for 1 hour and incubated at 4 ℃ overnight with the following primary antibodies: FOXP4 (1:1000, Abcam), GAPDH (1:10000, Abcam), MLL2 (1:1000, Abcam), E-cadherin (1:1000, Elabscience), vimentin (1:1000, Elabscience), and β-catenin (1:1000, Elabscience). Subsequently, the membranes were incubated with goat anti-rabbit IgG (KPL) for 1 hour, and then ECL reagent (Solarbio) was added for observation.
5
Protein Expression Analysis in EC Cells
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Total proteins were obtained from EC cells through radio-immunoprecipitation assay buffer (R0010; Solarbio). Next, the protein concentration was quantitated by the BCA kit (PC0020; Solarbio). Then, the proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto membranes (2215; Millipore, USA). After being blocked with 5% non-fat milk, membranes were reacted with primary antibodies against PCNA (ab92552, 29 kDa, ½,000), E-cadherin (ab231303, 97 kDa, 1 µg/mL), N-cadherin (ab18203, 100 kDa, 1 µg/mL), Vimentin (ab20346, 54 kDa, 1/1,000), VEGFR-1 (ab32152, 151 kDa, 1/2,000), VEGFR2 (ab11939, 151 kDa, 2 µg/mL), epidermal growth factor receptor (EGFR) (ab52894, 175 kDa, 1/2,000), and GAPDH (ab181602, 36 kDa, 1/10,000). Subsequently, the membranes were incubated with the appropriate secondary antibodies goat anti-rabbit IgG (ab205718, 1/5,000) as well as goat anti-mouse IgG (ab205719, 1/5,000), followed by being exposed to the Enhanced Chemiluminescence Substrate (PE0010; Solarbio) for visualization. All the antibodies were bought from Abcam (USA).
6
Sensitive Immunoassay Development Protocol
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1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enrofloxacin (ENR), ofloxacin (OFL), lomefloxacin (LOM), N,N-dimethylformamide (DMF), bovine serum albumin (BSA), HAuCl4·4H2O, citrate sodium, and ovalbumin (OVA) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). goat anti-rabbit IgG-HRP, goat anti-rabbit IgG, goat anti-mouse IgG-HRP, goat anti-mouse IgG, 3,3′,5,5′-tetramethylbenzidine solution (TMB), protein-A affinity column, and Tween- 20 were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Rabbit anti-ENR polyclonal antibody, mouse anti-OFL polyclonal antibody, mouse anti-LOM polyclonal antibody, and mouse anti-amide group polyclonal antibody were prepared by our laboratory in the previous studies (The immunogens of these antibodies were respectively ENR-cOVA, OFL-cOVA, LOM-cOVA, and cOVA, the titers of antisera were all over than 200,000). Monoclonal antibody specific to ENR was purchased from Wuhan Huamei Tech. Co., Ltd. (Wuhan, China). All other reagents were analytical or higher grade.
Detailed formulas of all solutions are given in Supporting Information.
Detailed formulas of all solutions are given in Supporting Information.
7
Western Blotting Protein Analysis
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Western blotting was performed as described previously.14 (link),15 (link) The primary antibodies used were rabbit anti-CSRP2 (Sigma, 1:500), rabbit anti-p21 (Cell Signaling Technology [CST], Beverly, MA, USA; 1:1000), rabbit anti-p27 (CST, 1:1000), rabbit anti-CDK4 (CST, 1:1000), rabbit anti-p-CREB (CST, 1:1000), rabbit anti-p-ERK (CST, 1:1000), rabbit anti p-P38 (CST, 1:1000), rabbit anti-p-SMAD3 (CST, 1:1000), rabbit anti p-AKT (CST, 1:1000), rabbit anti p53 (CST, 1:1000) and mouse anti-GAPDH (Solarbio, Beijing, China, 1:1000). The secondary antibody was goat anti-mouse IgG (Solarbio, 1:1000) and goat anti-rabbit IgG (Solarbio, 1:1000). The protein bands were determined using the ultra ECL kit (Biomed, Beijing, China) according to the manufacturer’s protocol.
8
Western Blot Analysis of HUVEC Proteins
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Total HUVEC protein was extracted using RIPA Lysis Buffer containing protease and phosphatase inhibitors. Protein concentration was measured using the BCA protein assay kit (Solarbio, Beijing, China). Total proteins were separated in 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Primary antibodies and corresponding dilutions were as follows: AKT (1:10000, Abcam, ab179463), p-AKT (1:5000, Abcam, Ab81283), eNOS (1:1000, Abcam, ab199956), p-eNOS (1:1000, Abcam, ab215717), PI3K (1:1000, Abcam, ab191606), p-PI3K (1:1000, #17366, CST). Secondary antibodies and corresponding dilutions were as follows: Goat Anti-Mouse IgG (1:5000, Solarbio, SE131), Goat Anti-rabbit IgG (1:5000, Solarbio, SE134). β-actin (1:1000, Solarbio, K200058M) was used as an internal control. Images are collected using Amersham Imager 600 (GE, CT, USA) and analyzed by ImageJ software. The experiment was repeated three times.
9
Western Blot Profiling of Protein Expression
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Proteins were extracted by mixing RIPA lysate (R0010, Solarbio) and PMSF (P0100, Solarbio). The concentrations of proteins were assayed by the BCA kit (PC0020, Solarbio). According to different molecular weights, 5% concentrated gel and 8% separation gel concentrations were used in the SDS-PAGE. After transferred to a PVDF membrane, the proteins were blocked by prepared 5% (M/V) BSA (Biosharp, BS043, China) in TBST buffer for 1 h and incubated overnight at 4 °C with the following primary antibodies: α-SMA (1: 500), COL-I (1: 500, AF0134, Affinity), TGF-β1 (1: 1000, AF1027, Affinity), proliferating cell nuclear antigen (PCNA) (1: 500, A12427, Abclonal, China), p-MEK1/2 (1: 500, AP0209, Abclonal), MEK1/2 (1: 500, A4868, Abclonal), p-ERK1/2 (1: 500, AF1015, Affinity), ERK1/2 (1: 500, AF6240, Affinity) and GAPDH (1: 10000, 60004-1-Ig, Proteintech, China). Next, the membrane was incubated with goat anti-mouse IgG (1: 3000, SE131, Solarbio) or goat anti-rabbit IgG (1: 3000, SE134, Solarbio) secondary antibody for 40 min at 37 °C. At last, the specific protein bands were visualized with Western ECL Substrate (D1010, Solarbio).
10
Quantitative Western Blot Analysis of AKR1C4
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Total proteins were extracted using Radio Immunoprecipitation Assay lysis buffer (Beyotime, China). Proteins (20µg/lane) from each tissue sample were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% (w/v) skimmed milk powder and immunoblotted with suitably diluted primary antibody AKR1C4 (Cat No: A7430, ABclonal, China) at a dilution of 1:200 followed by secondary antibodies (goat anti-rabbit IgG; Cat No: SE134, Solarbio, China) at dilution of 1:500 conjugated with horse radish peroxidase. Bands were visualized using enhanced chemiluminescence detection reagents (Thermo, USA) applied to Chemiluminescent Imaging System (Tanon, China). The quantification of western blot bands was carried out using ImageJ software v2.1 and normalized to GAPDH.
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