The tissues and cells were homogenized by using RIPA buffer that contains antiprotease mixture (Thermo, USA) to obtain total protein. The protein samples were quantified with a BCA kit (Beyotime, China), resolved in SDS-PAGE, shifted into NC membranes, blocked with 5% skim milk at room temperature for 1 hour, and hatched with primary antibodies against STAT3, phosphorylated STAT3, and β-actin (Proteintech, China, 1 : 500) at 4°C overnight. The next day, the membranes were incubated with HRP-conjugated, antimouse, or antirabbit secondary antibodies, visualized by an ECL substrate (Millipore, USA) on a gel image system (Bio-Rad, USA).
Primary antibodies against
Manufactured by Proteintech
Sourced in China
Primary antibodies are immunoglobulins that recognize and bind to specific target proteins or epitopes. They are a fundamental tool in immunodetection and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Hyperfilm, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Pvdf membrane, by Roche (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Antiprotease mixture, by Thermo Fisher Scientific (1 mentions)
Gel image system, by Bio-Rad (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Santa Cruz Biotechnology (1 mentions)
Anti rabbit secondary antibody, by Proteintech (1 mentions)
Enhanced chemiluminescence substrate kit, by Cytiva (1 mentions)
Pvdf membrane, by Proteintech (1 mentions)
Bca protein detection kit, by Beyotime (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Laser confocal microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Multiimager, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
11 protocols using primary antibodies against
1
STAT3 Phosphorylation Immunoblotting
2
Ovarian Cancer Protein Expression Analysis
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Ovarian cancer cells or tissues were lysed in radio‐immunoprecipitation assay buffer with protease inhibitors overnight, and the denatured protein samples were subjected to 10% or 12% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and electro‐transferred to PVDF membranes. Membranes were blocked in 3% bovine albumin serum (BSA) for 2 hours at room temperature and incubated with primary antibodies against β‐actin (1:5000) and VEGFA, TGF‐α and survivin (1:1000; Proteintech) overnight at 4°C, followed by anti‐rabbit secondary antibody (1:5000; Proteintech) for 2 hours, and then, protein bands were washed 3 times with PBS and then visualized using an enhanced chemiluminescence system (ECL, Santa Cruz Biotechnology).
3
SIRT1 Protein Expression Quantification
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The total protein extracted by RIPA buffer was subsequently quantified with BCA protein detection kit (Beyotime, Shanghai, China). The protein samples in different groups, were, respectively, mixed with the loading buffer, and then immediately heated in boiling water for 10 min. Then, the proteins were dissolved by SDS-PAGE and accordingly transferred onto the PVDF membrane (Beyotime, Shanghai, China), which was then blocked in 5% skimmed milk at ambient temperature for 2 h. Following that, the primary antibodies against SIRT1 and GAPDH (Proteintech, Wuhan, China) were used to incubate the PVDF membranes at 4°C for 12 h. Rinsed for three times with TBST buffer, the membranes were immediately incubated with secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature. The membranes were then rinsed three times with TBST buffer again, and an enhanced chemiluminescence substrate kit (Amersham Pharmacia Biotech, Little Chalfont, UK) was added onto the membranes, and the protein bands were developed.
4
Immunofluorescence Analysis of Renal CaSR
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Renal sections were fixed with a 4% paraformaldehyde solution for 4–6 hours at 4 °C, then transferred into a 30% sucrose solution overnight. Samples were embedded in optimal cutting temperature (OCT) compound, cut into serial 8 µm thick sections, placed on glass slides, and fixed in acetone for 10 minutes. Subsequently, slides were washed three times in phosphate-buffered saline (PBS) and blocked with 5% goat serum for 30 minutes at room temperature, followed by overnight incubation at 4 °C with primary antibodies against the CaSR (1:100, Proteintech Group). Slides were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:1,000, CST) for 1 hour at room temperature in the dark. After treatment with DAPI (Beyotime, Shanghai, China), the sections were washed an additional three times. Slides were then visualized with a laser confocal microscope (Olympus, Tokyo, Japan). The CaSR and nucleus were stained green and blue, respectively.
5
Exosome and Protein Expression Analysis
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Total proteins were extracted from PC cells using RIPA reagent containing 5% PMSF protease inhibitor (Boster, Wuhan, China). The BCA method was used to estimate protein concentrations. Proteins (30 μg per sample) were separated by 10% SDS-PAGE for 120 min and then transferred onto 0.45 μm pore size PVDF membranes (Millipore, USA). The membranes were then blocked using 5% non-fat milk and incubated for 12 h at 4° C with primary antibodies against PTEN, AKT, p-AKT, and β-actin (all from Proteintech, Wuhan, China). High sensitivity ECL reagent was used to visualize the blots in a Multi Imager (Bio-Rad, USA). Relative protein expression was calculated using ImageJ (NIH, USA). β-actin and AKT were used as references for PTEN and p-AKT, respectively. Antibodies against CD9, ALIX, and TSG101 (all from Proteintech, Wuhan, China) were used to positively identify exosomes.
6
Immunohistochemical Analysis of KIF11 Expression
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For IHC staining, the paraffin sections were deparaffinized, rehydrated, immersed in 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase, and then incubated for 30 min in blocking buffer containing 5% bovine serum albumin. After blocking, the sections were incubated with primary antibodies against KIF11 (1:100; Proteintech, China) overnight at 4 ℃, followed by incubation at room temperature with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:100; Sigma-Aldrich, USA). The IHC staining results for the KIF11 proteins were determined using a semi-quantitative analysis technique, in which samples were analyzed using a bright-field microscope (Nikon, Japan). For each sample, the protein expression intensity was determined using ImageJ software. The KIF11 values were expressed as the percentage of positive cells in each case. The cell staining intensity was divided into two categories: cases with greater than or equal to 30% positive nuclei were classified as KIF11-high group, and those with less than 30% were classified as KIF11-low group. Three independent observers inspected the specimens in a blinded manner.
7
Anti-inflammatory Mechanisms of Brevilin A
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Brevilin A (BVA) was provided by PUSH Bio-Technology (Chengdu, China, Figure 1A , the purity ≥98.0%). LPS (E. coli 0111:B4) was purchased from Sigma (Shanghai, China). Murine IFNγ and TNFα were obtained from SinoBiological (Beijing, China). Curcumin, BAY 11-7082, MG132, and LY294002 were purchased from Beyotime (Shanghai, China). PD98059, SB203580, and SP600125 were provided by Selleck (Shanghai, China). Primary antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), poly [ADP-ribose] polymerase 1 (PARP1), GAPDH, and Tubulin were obtained from ProteinTech (Wuhan, China). The antibodies against IKKα/β, phosphorylated-IKKα/β, IκBα, phosphorylated-IκBα, NF-κB p65, and Flag were purchased from Beyotime (Shanghai, China). Extracellular regulated protein kinases (ERK), phosphorylated-ERK, c-Jun N-terminal kinases (JNK), phosphorylated-JNK, p38, phosphorylated-p38, RAC-alpha serine/threonine-protein kinases (Akt), and phosphorylated-Akt were provided by Signalway Antibody (Baltimore, United States).
8
Immunohistochemical Analysis of Disc Degeneration
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ACAN, Bax, and Beclin1 expression levels were detected using immunohistochemical staining. Briefly, paraffin slices of intervertebral disc tissue were heated for 2 h at 60°C, dewaxed in xylene for 30 min, dehydrated in graded ethanol for 5 s, and blocked with 3% H2O2 for 10 min. Slices were then washed in PBS for 15 min, incubated with 0.4% pepsin in 5 mM HCL for antigen retrieval for 30 min at 37°C, and blocked with goat serum for 30 min at room temperature. Next, the slices were incubated with primary antibodies against ACAN (1 : 200, Proteintech, China), Bax, (1 : 400, Proteintech), and Beclin1 (1 : 200, CST, USA) overnight at 4°C. The next day, the slices were washed three times with PBS for 15 min and incubated with HRP-conjugated secondary antibody for 2 h at room temperature. The slices were stained with 3, 3-diaminobenzidine, counterstained with hematoxylin, dehydrated with graded ethanol, and analyzed by light microscopy (Leica, Germany).
9
Western Blot Analysis of Aortic Tissues
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Aortic tissues were lysed by RIPA Lysis Buffer (Solarbio, Beijing, China) and protein concentration was measured by BCA protein assay kit (Solarbio, Beijing, China). The prepared protein was separated by polyacrylamide-SDS gels and then transferred onto PVDF membranes (Roche, Switzerland). Blocked with 5% skimmed milk for 2.5 h, the PVDF membrane was subjected to incubation with primary antibodies against VEGF, Hsp70 and β-actin (1:1000, Proteintech Group Inc., Wuhan, China) at 4°C overnight. On the following day, protein samples were incubated with the secondary antibody at 37°C for 45 min. The protein blots were visualized using enhanced chemiluminescence (ECL) with exposure to X-ray films (Hyperfilm, GE Healthcare, UK, USA). β-actin was used as internal control, and the gray value ratio of the protein band to β-actin was deemed as relative protein expression.
10
Protein Expression Analysis in Aortic Tissues
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Aortic tissues were lysed by RIPA Lysis Buffer (Solarbio, Beijing, China), and protein concentration was measured by the BCA protein assay kit (Solarbio, Beijing, China). The prepared protein was separated by polyacrylamide-SDS gels and then transferred onto PVDF membranes (Roche, Switzerland). Blocked with 5% skimmed milk for 2.5 h, the PVDF membrane was subjected to incubation with primary antibodies against VEGF, Hsp70 and β-actin (1:1000, Proteintech Group Inc., Wuhan, China) at 4°C overnight. On the following day, protein samples were incubated with the secondary antibody at 37°C for 45 min. The protein blots were visualized using the enhanced chemiluminescence (ECL) with exposure to X-ray films (Hyperfilm, GE Healthcare, UK, USA). β-actin was used as internal control, and the gray value ratio of the protein band to βactin was deemed as relative protein expression.
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