Aortic valve tissue and human U937 macrophages were lysed using RIPA lysis buffer, and total proteins were obtained and adjusted to the same concentration. Total proteins were added to a 10% SDS polyacrylamide gel to electrophoretically separate proteins by their different molecular weights, and all proteins were then transferred to Immobilon-FL PVDF membranes. After being blocked with 3% BSA for 90 minutes at room temperature, the membranes were incubated with primary antibodies for 2 hours at 37°C and with secondary antibodies for 1 hour at room temperature. Then, the expression of target proteins was examined by Odyssey. The target proteins in aortic valve tissue were Sesn2 (GeneTex, Cat No.: GTX116925, 1:1500) and GAPDH (GeneTex, Cat No.: GTX100118, 1:5000), and the target proteins in human U937 monocytes were Nrf2 (GeneTex, Cat No.: GTX103322, 1:1500) and GAPDH.
Gapdh
Manufactured by GeneTex
Sourced in United States, United Kingdom, China
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a housekeeping gene that encodes an enzyme involved in glycolysis, the metabolic pathway that converts glucose into energy. The GAPDH protein is commonly used as a control or reference in various biological experiments due to its consistent expression across different cell types and experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (14 mentions)
β actin, by Merck Group (14 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Bcl 2, by GeneTex (7 mentions)
C myc, by Abcam (7 mentions)
Flag tag (flag), by Merck Group (7 mentions)
β actin, by Santa Cruz Biotechnology (7 mentions)
Vimentin, by GeneTex (6 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Caspase 3, by Cell Signaling Technology (6 mentions)
P erk, by Cell Signaling Technology (6 mentions)
β actin, by GeneTex (5 mentions)
Bcl2 associated x (bax), by GeneTex (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Ampkα, by Cell Signaling Technology (4 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
Bcl 2, by Cell Signaling Technology (4 mentions)
N cadherin, by GeneTex (4 mentions)
β tubulin, by Santa Cruz Biotechnology (4 mentions)
204 protocols using gapdh
1
Protein Expression Analysis in Aortic Valve and Macrophages
2
Probing CVA10 Interactions with hSCARB2
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Cell lysates (50 µg) from RD cells that had been infected with a multiplicity of infection (MOI) = 0.05 of CVA10 and cultured on 10 cm dishes for 24 h were mixed with 5 μL of protein G magnetic beads (Cytiva, Washington, DC, USA) and 1 µL of pull-down rabbit antibody specific to CVA6/CVA10, hSCARB2 (NOVUS Biologicals, Littleton, CO, USA), KRM1 (GeneTex, Inc.), or isotype antibody, which is specific to GAPDH (GeneTex, Inc.), respectively, at 4 °C for 6 h. After five washes with lysis buffer, lysate–magnetic beads were resuspended in 30 μL SDS loading buffer, boiled for 5 min, and then subjected to SDS-PAGE and Western blotting with primary antibody reacting to CVA/CVA10, hSCARB2, KRM1, or GAPDH. Detection with anti-hSCARB2 primary antibody (R&D, AF1966) used anti-goat IgG conjugated with HRP. The other detection was achieved using an appropriate anti-rabbit antibody conjugated with HRP (GeneTex, Inc., Rabbit EasyBlot kit, GTX225856-01). Cell lysates from 3T3-SCARB2 cells in the presence or absence of CVA10 infection were directly subjected to Western blotting, as described above. Each experiment has been repeated at least two times independently.
3
Protein Expression Analysis of IL-22 Signaling in Human Atrial Tissue
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Human atrial tissue was dissolved in RIPA buffer [50 mM; Tris (pH 7.4), 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM odglycerophosphate, 1 mM EDTA, 1 mM Na3VO4, 0.5 µ/ml leupeptin 25˚C] and further lysed at 5,000 Hz using an Ultrasonic Lysimeter (model, XL-2000; Misonix). Subsequently, the atrial tissue homogenate was centrifuged at 6,000 x g at 4˚C for 15 min. The supernatant of each sample was collected and total protein concentration was detected using a bicinchoninic acid Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 30 µg protein with different molecular weights were separated via electrophoresis on a 10% gel. After transferring the separated proteins to Immobilon-FL PVDF membranes (EMD Millipore) at 200 mA for 1 h, the blots were blocked with 5% nonfat milk (25˚C, 1 h) and incubated with antibodies against IL-22R1 (1:1,000; cat. no. GTX16978; GeneTex, Inc.), IL-10R2 (1:1,000; cat. no. GTX00131-pro; GeneTex, Inc.), IL-22 (1:1,000; cat. no. GTX109659; GeneTex, Inc.) and GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.) at 4˚C for 10 h. Following incubation with goat anti-rabbit IgG H&L Alexa Fluor 647 secondary antibody (1:10,000; cat. no. 150079; Abcam) at 25˚C for 1 h, the blots were scanned using the Odyssey Imaging System (LI-COR Biosciences).
4
Western Blot Protein Analysis
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Cell samples were transferred to a microcentrifuge tube and lysed using Tissue Cell Lysis Buffer (CAT No: GB-181-100). After centrifugation at 20,000 × g for 15 min and protein quantification, SDS-PAGE was carried out using 30 μg of protein in each sample. Next, proteins were transferred onto PVDF membranes (Merck Millipore) and blocked for 1 h at room temperature using PBS containing 5% bovine serum albumin (BSA). Antibodies used for western blotting were total pERK1/2 (Thr202/Tyr204; D13.14.4E; Cell Signaling-4370 1:1,000), pAkt (Ser-473; D9E; Cell Signaling-4060 1:500), and GAPDH (GeneTex, 1:5,000). Primary antibodies were incubated overnight at 4°C and washed off. Species-specific HRP-conjugated secondary antibodies were then incubated for 1 h at room temperature and washed extensively. Finally, membranes were incubated with Super Signal West Femto substrate (Thermo Fisher Scientific), and signals were detected using the in vivo Xtreme imaging system (Bruker, Billerica, MA, United States).
5
Protein Expression Analysis in Xenografts
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Western blotting was performed to analyse protein expression. The antibodies used were specific for EIF4A3 (1:1000; Abcam) and RhoU (1:500; Origene). GAPDH (1:6000, GeneTex) and β-actin (1:3000, GeneTex) were used as the controls. Protein bands were visualized using an enhanced chemiluminescence (ECL) chromogenic substrate (Beyotime, Shanghai, China) and assessed by Image-Lab analysis software (San Leandro, CA, USA).
Paraffin-block tissues of subcutaneous xenografts in mice were stained with haematoxylin and eosin (HE) and IHC, and subsequently evaluated by a pathologist blindly.
Paraffin-block tissues of subcutaneous xenografts in mice were stained with haematoxylin and eosin (HE) and IHC, and subsequently evaluated by a pathologist blindly.
6
Proteomic Analysis of DNA Damage Response
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Cell lysates were preparing employing RIPA buffer (50mM Tris pH=7.4, 150mM NaCl, 2mM EDTA, 0.5% NP40, 0.25% Sodium Deoxycholate) supplemented with Complete protease inhibitor and PHOSStop phosphatase inhibitor (Roche). Hundred micrograms of protein were separated in 10% SDS PAGE and transferred to a PVDF membrane. The membrane was probed with antibodies for FOXM1, Cyclin B, CHK1 S345, (CST), Rad51, Exo1 (Abcam), RPA2 s4/s8 (Bethyl), GAPDH (GeneTex), tubulin (T6074, Sigma). Bound antibodies were detected with infrared fluorescent secondary antibodies and imaged with a LI-COR Odyssey system. The numbers below the bands are fold of change over the non-treated condition calculated from the relative intensity of the bands quantified employing Image Studio Software.
7
Western Blot Analysis of Cellular Proteins
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Protein lysates were generated from cells, scraped into RIPA buffer (Sigma-Aldrich, USA). Protein concentration was measured using BCA protein assay kit (Thermo Scientific, USA). Gel-electrophoresis was conducted on 4–12% NuPAGE gels (Invitrogen, USA) at 100V for 2hr. Proteins were transferred on 0.2 μm pore nitrocellulose membranes using iBlot gel transfer stacks (Invitrogen, USA), utilizing IBlot gel transfer device (Invitrogen, USA) at 20 V for 7min. Membranes were blocked with 2.5% milk (Bio-Rad, USA), diluted in TBS (Corning, USA) with 0.05% Tween 20 (Sigma-Aldrich, USA) for 1hr and incubated with following primary antibodies: anti-pSmad3 (Abcam, USA), anti-collagen I (Abcam, USA) and GAPDH (GeneTex, USA), diluted 1:7000 at 4°C overnight. Secondary HRP-conjugated anti-rabbit and anti-mouse antibodies (Promega, USA) were used in 1:30000 dilution for 2 hours at room temperature. Protein bands were detected with ECL reagent (Thermo Scientific, USA) on ChemiDoc imaging system (Bio-Rad, USA). Images were analyzed using ImageJ software (NIH).
8
Cytoplasmic and Nuclear Protein Isolation
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The cytoplasm and nuclei in LV tissue and RAW264.7 macrophages were isolated using a nuclear separation kit (Njjcbio) according to a previously described protocol [19 (link)]. Briefly, homogenate was collected from LV tissue and cells that were lysed in lysis buffer and further centrifuged at 1000 × g for 10 minutes. The pellets in the bottoms of the tubes contained the nuclei, and the supernatant contained the cytoplasm.
After the cytoplasm, nuclei, LV tissue, and HL-1 cardiomyocytes were, respectively, lysed in RIPA lysis buffer containing 10% protease inhibitors and 10% phosphatase inhibitors, the total protein was obtained and quantitated using a BCA Protein Assay Kit. After separation by electrophoresis on 10% SDS polyacrylamide gels, the proteins were transferred to PVDF membranes. Then, the protein expression of IL-16 (Abcam), Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH (all three from GeneTex) in total LV tissue, the protein expression of p-p65 and GAPDH (both from Abcam) in the cytoplasm, the protein expression of p-p65 and PCNA (GeneTex) in the nuclei, and the protein expression of Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH in HL-1 cardiomyocytes were measured using primary antibodies as indicated in parentheses. After further incubation with the secondary antibodies, the target proteins were detected and analyzed.
After the cytoplasm, nuclei, LV tissue, and HL-1 cardiomyocytes were, respectively, lysed in RIPA lysis buffer containing 10% protease inhibitors and 10% phosphatase inhibitors, the total protein was obtained and quantitated using a BCA Protein Assay Kit. After separation by electrophoresis on 10% SDS polyacrylamide gels, the proteins were transferred to PVDF membranes. Then, the protein expression of IL-16 (Abcam), Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH (all three from GeneTex) in total LV tissue, the protein expression of p-p65 and GAPDH (both from Abcam) in the cytoplasm, the protein expression of p-p65 and PCNA (GeneTex) in the nuclei, and the protein expression of Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH in HL-1 cardiomyocytes were measured using primary antibodies as indicated in parentheses. After further incubation with the secondary antibodies, the target proteins were detected and analyzed.
9
Analyzing Cell Signaling Pathways
10
Regulation of mTOR Signaling Pathway
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Primary antibodies specific to mTOR were purchased from Abcam (Cambridge, MA, USA). Akt, phospho-mTOR (Ser2448), phospho-Akt (Ser473), phospho-AMPKα (Thr172), AMPKα, cleaved caspase 3, and cleaved caspase 9 (Cell Signaling; Danvers, MA, USA); Bcl-XL, Bcl-2, Bax, Bak, poly ADP-ribose polymerase (PARP), p62, microtubule-associated proteins 1A/1B light chain 3B (LC3B), and GAPDH (Genetex; Irvine, CA, USA); and cyclin-dependent kinase 1 (CDK1) and cyclin B1 (Merck Millipore; Burlington, MA, USA). Anti-rabbit polyclonal and anti-mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). ROS scavengers N-acetylcysteine (NAC) and H2O2-scavenging enzyme catalase were purchased from Merck Millipore (Burlington, MA, USA). Pan-caspase inhibitor z-VAD-FMK was purchased from R&D Systems (Minneapolis, MN, USA). Naringenin (N5893) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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