Protein levels of PCBP2 and CDK2 were detected using western blotting, which was performed essentially as described previously 16 , 17 . PCBP2 rabbit polyclonal antibody, CDK2 mouse monoclonal antibody (both dilution 1 : 1000; Proteintech Group, Inc.) and GAPDH rabbit polyclonal antibody (dilution 1 : 10 000; Proteintech Group, Inc.) were used. GAPDH was examined as a control.
Gapdh rabbit polyclonal antibody
Manufactured by Proteintech
Sourced in China, United States
The GAPDH rabbit polyclonal antibody is a reagent used for the detection and quantification of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein in various biological samples. GAPDH is a key enzyme involved in the glycolytic pathway and is commonly used as a loading control or reference protein in Western blot and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ecl reagent, by Vazyme (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cdk2 mouse monoclonal antibody, by Proteintech (1 mentions)
Beta tubulin rabbit polyclonal antibody, by Proteintech (1 mentions)
Beta actin rabbit polyclonal antibody, by Proteintech (1 mentions)
Protease phosphatase inhibitor, by Merck Group (1 mentions)
Hif 1α mouse monoclonal antibody, by Novus Biologicals (1 mentions)
Alexa fluor 680 790 labeled goat anti rabbit goat anti mouse igg antibody, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Polycarbonate membrane, by Corning (1 mentions)
Chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Transwell system, by Corning (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
E cadherin rabbit polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Ccd 18co, by American Type Culture Collection (1 mentions)
10 protocols using gapdh rabbit polyclonal antibody
1
Quantifying PCBP2 and CDK2 Protein Levels
2
Western Blot Analysis of S100A11
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer according to the manufacturer’s instructions. The concentrations of the protein samples were determined using the bicinchoninic acid (BCA) assay method, and 50µg protein from each sample was subjected to 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). After the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, the membranes were rinsed with TBST and blocked with skimmed milk solution for 1 hour. After that, membranes were incubated overnight with S100A11 rabbit monoclonal antibody (1:1000; Cat. no. 0057513; Proteintech, Wuhan, China) or GAPDH rabbit polyclonal antibody (1:5000; Cat. no. 10494-1-AP; Proteintech, Wuhan, China) 4°C. After washing with TBST for 4 times, Membranes were incubated with goat anti-rabbit secondary antibody (1:2,000; Cat. no.ZB-2301, ZSJQ-BIO) for 1.5 hours at room temperature. Following washing with TBST for 4 times, the blots were developed using the enhanced chemiluminescence (ECL) reagent (Vazyme, Nanjing, China) and the images were recorded in the Gel Imaging System. The relative S100A11 expressions were calculated by the Gel-Pro-Analyzer (Tanon, Shanghai, China).
3
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from tissues and cells and lysed in RIPA buffer (Beyotime, China) mixed with protease/phosphatase inhibitors (Sigma-Aldrich, USA). Samples were collected via centrifugation at 13,000 g and 4 °C for 5 min. The supernatants were boiled at 100 °C for 5 min in loading buffer. A BCA protein assay (Thermo Scientific, USA) was used to measure protein concentrations. Equal amounts of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with primary antibody (HIF-1α mouse monoclonal antibody, 1:2000, Novus; TNF alpha rabbit polyclonal antibody, 1:1000, Abcam; MCP-1 mouse monoclonal antibody, 1:1000, Novus; beta tubulin rabbit polyclonal antibody, 1:1000, Proteintech; GAPDH rabbit polyclonal antibody, 1:1000, Proteintech; beta actin rabbit polyclonal antibody, 1:1000, Proteintech). An Alexa Fluor 680/790-labeled goat anti-rabbit/goat anti-mouse IgG antibody (1:25,000, LI-COR Biosciences, USA) was used as the secondary antibody. An Odyssey infrared imaging system (LI-COR Biotechnology, USA) was employed to visualize the blots.
4
Colorectal Cancer Cell Line Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human colon cancer cell lines HT-29 and SW-620 were purchased from the Cell Center of Xiangya School of Medicine, Central South University (Hunan, China). Human normal colon cell line CCD-18Co was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Both miR-133b mimics and inhibitor were synthesised by Shanghai GenePharma Co., Ltd (Shanghai, China). The transfection reagent Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). P63 rabbit monoclonal antibodies were purchased from Abcam (Cambridge, UK). RhoA rabbit polyclonal antibody, E-cadherin rabbit polyclonal antibody and vimentin rabbit polyclonal antibody were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH rabbit polyclonal antibody was purchased from Proteintech Group, Inc. (Chicago, IL, USA). The Alexa Fluor 488 Goat Anti-Rabbit IgG used for immunofluorescence was purchased from Invitrogen. The Chromatin Immunoprecipitation Kit was purchased from EMD Millipore Corporation (Billerica, MA, USA). A transwell system (24 wells, 8 μm pore size with poly-carbonate membrane) was purchased from Corning Costar (Tewksbury, MA, USA). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA).
5
Evaluation of HepaRG Cells for Drug Metabolism
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepaRG cell lines were supplied by Guandao Biological Engineering (Shanghai, China). RPMI 1640 medium and FBS were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Trypsin–EDTA 0.25% solution and 1% antibiotics (100 × streptomycin–penicillin) were purchased from Biosharp (Shanghai, China) and HyClone (Logan, UT, USA), respectively. SsD (S114050, 20 mg, ≥98% purity), phenacetin, and dextromethorphan hydrobromide were purchased from Aladdin Bio-Chem Technology (Shanghai, China). MTT, PBS, and DMSO were provided by Solarbio Technology (Beijing, China). GAPDH rabbit polyclonal antibody, CYP1A2-specific rabbit polyclonal antibody, HRP-conjugated AffiniPure goat antirabbit IgG (H+L), and CYP2D6 rabbit polyclonal antibody were obtained from Proteintech (Wuhan, China).
6
Osteoblast Differentiation Marker Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein expression of osteoblast differentiation markers was determined by western blot analysis. Briefly, cells were lysed using M-PER mammalian protein extraction reagent containing a protease inhibitor (Thermo Fisher Scientific, USA). And protein concentrations were tested with Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s specifications. Then, the lysates were separated on an 8% SDS/PAGE. After electrophoretic transfer on to nitrocellulose membranes (Thermo Fisher Scientific) and blocking with 5% milk solution, blots were incubated overnight at 4 °C with primary antibodies including anti-Runx2 rabbit monoclonal antibody (1:2000, Epitomics, CA), anti-Sp7/Osterix rabbit polyclonal antibody (1:1000, Abcam, UK), and GAPDH Rabbit Polyclonal Antibody (1:5000, Proteintech, China). Then, they were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000, Jackson, USA). The protein bands were detected and visualized by the imaging system (Tanon 5500, China) after being incubated with the SuperSignal™ West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific). Densitometry analyses of the western bands were performed using the ImageJ Imaging software.
7
Western Blot Analysis of S100A10 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This part refers to our previous experimental methods20 (link). Briefly, Total cellular proteins were lysed with a radio-immunoprecipitation assay (RIPA). The concentrations of the proteins were determined using the bicinchoninic acid (BCA) assay method and equal concentrations of the proteins were loaded on SDS-PAGE gel (10%). Thereafter, the samples were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk solution for 1 hour. Next, membrane was incubated with S100A10 rabbit monoclonal antibody (1:1000; Cat. no. 0041696; Proteintech, Wuhan, China) or GAPDH rabbit polyclonal antibody (1:5000; Cat. no. 10494-1-AP; Proteintech, Wuhan, China) at 4°C overnight. After washing with PBST for 4 times, the membranes were incubated with horseradish peroxidase-linked secondary biotinylated antibodies for 1.5h at room temperature. Following washing with PBST for 4 times, the immunoreactive bands were observed using the enhanced chemiluminescence (ECL) reagent (Vazyme, Nanjing, China), and the images were recorded in the Gel Imaging System. The relative S100A10 expression levels were calculated by the Gel-Pro-Analyzer (Tanon, Shanghai, China).
8
Western Blot Analysis of IGF2BP2 and IGF2BP3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Utilized RIPA lysis buffer with 1% phenylmethanesulfonyl fluoride (PMSF) and DL-dithiothreitol (DTT) to extracted total protein. The BCA protein assay kit (Beyotime Biotechnology) was used to determine the concentration of the protein lysate. Equivalent (30 μg) protein was isolated by 10% SDS-PAGE. Then, the proteins were transferred to PVDF membranes (0.45 mm; Beijing Solarbio Science & Technology Co., China). Before incubated the membranes with IGF2BP2 and IGF2BP3 antibodies (1:1000, R&D Systems, MN, USA) at 4 °C for 12 h, the membranes were blocked at room temperature with 5% BSA for 1 h. Then GAPDH rabbit polyclonal antibody (1:4000, Proteintech, USA) was utilized as a loading control for normalization. HRP-conjugated secondary anti-rabbit antibody (1:4000; ProteinTech Group) was incubated at room temperature about 1 h. Finally, the bands were placed on an Omega Lum G machine (Aplegen, USA) and visualized using ECL reagents (Thermo Fisher Scientific).
9
Murine Pancreatitis Model Exploration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Male C57BL/6 J mice from Beijing Vital River (China) were used in this study. The AR42J cell line and F-12 K medium were obtained from the BeNa culture collection (China). Recombinant mouse Interleukin -22 (Miltenyi, Germany) was used, along with Caerulein (Sigma, America) and Lipopolysaccharide (Solarbio, China). AKT, mTOR, anti-mTOR (phosphor s2448), and anti-AKT (Ser473) antibodies were purchased from Proteintech(China). LC3 and P62 antibodies were obtained from Abclonal(China), and GAPDH Rabbit Polyclonal antibody and goat anti-rabbit IgG(H + L) HRP conjugate were from Proteintech(China). Trizol reagent, PCR primers, Reverse transcription kit, and SYBR Green qPCR kit were purchased from Agbio(China).
10
Quantitative Protein Analysis in MDMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from MDMs using RIPA lysis buffer (Cell Signaling, Danvers) plus protease and phosphatase inhibitors. The protein concentration of each sample was measured using a standard Bradford assay (BioRad, Hercules) and equal amounts of protein were loaded onto a 12% SDS polyacrylamide gel. After gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane using a wet-transfer system, and the membrane was blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% nonfat dry milk. The membrane was immunoblotted overnight at 4°C with AAT rabbit polyclonal antibody (DAKO, Carpinteria) at a dilution of 1:5,000 in TBST. horseradish peroxidase conjugated anti-rabbit antibody (BioRad, Hercules) was used for secondary labeling at 1:5,000 in TBST for 1 h at room temperature. The membrane was reprobed with GAPDH rabbit polyclonal antibody (Proteintech, Rosemont) at 1:5,000 in TBST. A horseradish peroxidase conjugated anti-rabbit (BioRad, Hercules) was used for secondary labeling. Protein bands were visualized by enhanced chemiluminescence (ECL, GE Healthcare, Chicago).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!