Proteins were extracted from rat ovarian granulosa cells, washed with HBSS and then lysed with RIPA buffer (Sigma-Aldrich, USA) containing protease inhibitors (Roche, Germany) for 30 min. The Bradford assay was used to determine the protein concentration. Equal amounts of total protein (15 μg) were loaded, separated on SDS-PAGE, transferred to a nitrocellulose membrane, and then immunoblotted with the appropriate antibody: ERα (#04-820, Millipore), ERβ (#92731, Millipore), FSHR (#sc13935; Santa Cruz, CA) StAR (#sc25806, Santa Cruz, CA), aromatase (#14245, Santa Cruz, CA), β-actin (13E5, Cell Signaling Technology), GADPH (#sc48116, cell signaling). ECL detection kit (GE, health care) was used to visualize the protein and Quality One software (Bio-Rad) was applied to quantify the intensities.
Gadph
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
GAPDH is a common internal control protein used in western blotting and other protein analysis techniques. It is a key enzyme involved in glycolysis, the metabolic pathway that converts glucose to energy. GAPDH is a widely expressed and constitutively active protein, making it a useful reference for normalizing protein levels between samples.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Cell Signaling Technology (8 mentions)
Caspase 3, by Cell Signaling Technology (6 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (6 mentions)
Cleaved caspase 9, by Cell Signaling Technology (5 mentions)
Caspase 9, by Cell Signaling Technology (5 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
β catenin, by Cell Signaling Technology (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
P akt, by Cell Signaling Technology (4 mentions)
P mtor, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nf κb p65, by Cell Signaling Technology (3 mentions)
E cadherin, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
76 protocols using gadph
1
Ovarian Granulosa Cells Protein Analysis
2
Antibody-based Analysis of ALK Signaling
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Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA). Pan-ERK1/2 (1:2000; 610123), was purchased from BD Transduction Laboratories (Franklin Lakes, NJ). Ki67 was from BD Pharmingen, (1:100; 558616). Monoclonal antibody 135 (anti-ALK) was described (Chand et al., 2013 (link); Moog-Lutz et al., 2005 (link)). Horseradish-peroxidase-conjugated secondary antibodies goat anti-rabbit IgG (PIEA31460) and goat anti-mouse IgG (PIEA31394; both at 1:5000) were from Thermo Scientific (Waltham, MA).
Crizotinib was from Haoyuan Chemexpress Co., Limited, Shanghai, China. PF-04643922 was from Pfizer, California, USA.
Crizotinib was from Haoyuan Chemexpress Co., Limited, Shanghai, China. PF-04643922 was from Pfizer, California, USA.
3
Western Blot Analysis of Protein Expression
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Tumor samples were solubilized in radioimmunoprecipitation assay (RIPA) buffer for 30 minutes at 4°C with shaking for protein extraction and the protein was quantitated by the BCA protein assay (Invitrogen, CA, USA). Equal amounts of protein (40 μg) from the different groups were subjected to electrophoresis on a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel, then transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA for 2 hours, then incubated at 4°C overnight with primary antibodies PNO1 (1: 800, catalog numbers: ab163419) (Abcam, Cambridge, United Kingdom) or AKT, mTOR, COX2 (1: 1000, Cell Signaling Technology, MA, USA) and GADPH (1: 2000, Cell Signaling Technology, MA, USA). GAPDH was used as the loading control. Membranes were washed 3 times with PBS (5 minutes each), then incubated with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (1: 10000, catalog numbers: sc2357) (Santa Cruz Biotechnology, CA, USA). Membranes were visualized by chemiluminescence and using Image Lab software 4.1 (Bio-Rad, CA, USA) for densitometry analysis.
4
Telomerase Immortalized hMEC Signaling
5
Comprehensive Molecular Profiling of Cells
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Western blotting and Immunohistochemically staining were performed as previously described [35 (link)]. The primary antibodies used in WB concludes of ESR1 (ab108398, 1:2000, Abcam), cyclin d1(#2978, 1:2000, Cell Signaling Technology), DICER1 (#3363, 1:2000, Cell Signaling Technology), NICD (#2421, 1:1000, Cell Signaling Technology), Vinculin (#13901, 1:5000, Cell Signaling Technology), NUMB (ab14140, 1:2000, Abcam), GADPH (#5174, 1:5000, Cell Signaling Technology). For immunobiological assay, the slides were incubated with anti-CD44 antibody (#3570, 1:500, Cell Signaling Technology), ALDH1 antibody (#54135, 1:500, Cell Signaling Technology) and NUMB antibody (ab14140, 1:1000, Abcam).
6
Western Blot Analysis of Cell Signaling Proteins
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The protocol of western blot assay were performed as reported previous [11 (link)]. The dilution of RABL6 (No.400055684-A01), E-cadherin (ab15148), β-catenin (ab16051), Vimentin (ab8978) and slug (ab106077) used for western blot was 1:1000, while the dilution of a-tubulin and GADPH was 1:5000. β–catenin, slug, a-tubulin (ab7291), GADPH (ab181602), E-cadherin and Vimentin was applied by Cell Signaling Technology (Beverly, MA, United States). GADPH and a-tubulin was probed on the membranes as an internal control antibody, for the sake of confirming equal loading.
7
Protein Expression Analysis in Renal Tissues
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Renal tissues were washed by PBS and lysed in lysis buffer (Beyotime, Shanghai, China). The lysates were then incubated on ice for 30 min and oscillated for 30 s. The cell lysates were centrifuged at 10,000 g for 30 min at 4°C. The supernatant was collected to measure the protein concentrations by BCA kit (Solarbio, Beijing, China). The proteins separated by the SDS-PAGE were transferred onto polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK). After being blocked for 1 h, the membranes were incubated overnight at 4°C with primary antibodies as follows: Bcl-2 (1:500, Abcam, Cambridge, UK), Bax (1:500, Abcam), caspase-3 (1:500, Abcam), HIF-1α (1:1000, Abcam), VEGF (1:1000, Abcam), survivin (1:1000, Cell Signaling Technology), p21 (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology) and GADPH (1:1000, Cell Signaling Technology). Thereafter, the membranes were probed with secondary antibodies (1:1000, Beyotime). The bands were determined by a Molecular Imager VersaDoc MP 5000 System (Bio-Rad, Hercules, CA). The densitometry was determined with a Quantity One (Bio-Rad).
8
Western Blot Analysis of DNA Damage Response
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Western blotting was performed as previously described [49 (link)]. Briefly, equal amounts of lysates were resolved with SDS-PAGE and were transferred onto PVDF membranes. Membranes were incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Signal amplification and detection were achieved by exposing the membrane to enhanced chemiluminescence reagent (GE Healthcare, Buckinghamshire, UK), followed by visualization using the Storm imaging system (Amersham Biosciences, Piscataway, NJ, USA). The following primary antibodies were used: γ-H2AX (1/1000; Cell Signaling Technology, Beverly, MA, USA), Chk2 (1/500; Cell Signaling Technology), Phospho-Chk2 (Thr68) (1/1000; Cell Signaling Technology), XRCC2 (1:1500; Abcam), Caspase-9 (1/500; Proteintech, Chicago, IL, USA), Caspase-3 (1/500; Proteintech), PARP (1/500; Proteintech), and BCL-2 (1/500; Santa Cruz Biotechnology). Detection of GADPH (1/10000; Cell Signaling Technology) was used as a loading control. Bound antibodies were visualized with peroxidase-linked secondary antibodies (anti-rabbit antibody: 1/10000; Cell Signaling Technology and anti-mouse antibody: 1/5000; Sigma-Aldrich, St. Louis, MO, USA).
9
Cultivation and Characterization of Morel Mushroom
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Fruiting bodies of Morchella importuna were obtained from fields owned by the Sichuan Academy of Agricultural Sciences (SAAS). Molecular sequences generated from the fruiting bodies (no. Cyl-158) were deposited at GenBank (http://www.ncbi.nlm.nih.gov ) under accession numbers MG121861–MG121865. The fruiting bodies were dried in a 37°C oven. The Sephadex 30 increase column was obtained from GE Healthcare (Uppsala, Sweden). The Brownlee analytical C18 column was bought from Perkin Elmer (Shelton, USA).
The human cervical cancer HeLa cell line was acquired from the West China Medical College, Sichuan University. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA were from Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO). Kits utilized to determine caspase activity (caspase-3 and -9) were obtained from Beyotime (Shanghai, China). Antibodies for Bax, Bcl-2, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and GADPH were from Cell Signaling Technology (Danvers, MA, USA). Other chemical reagents involved in this research were of analytical grade.
The human cervical cancer HeLa cell line was acquired from the West China Medical College, Sichuan University. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA were from Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO). Kits utilized to determine caspase activity (caspase-3 and -9) were obtained from Beyotime (Shanghai, China). Antibodies for Bax, Bcl-2, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and GADPH were from Cell Signaling Technology (Danvers, MA, USA). Other chemical reagents involved in this research were of analytical grade.
10
Targeted BTK Degradation by Bifunctional Inhibitors
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Example 3
K562 cells were treated with DMSO or with 200 nM, 1 μM, or 5 μM of a BTK bifunctional inhibitor compound of the disclosure for 12 hours, followed by protein lysate harvest and Western blotting analysis. As shown in
MOLM14 cells were treated with DMSO or with 40 nM, 200 nM, 1 μM of a BTK bifunctional inhibitor compound of the disclosure for 4 hours, followed by protein lysate harvest and Western blotting analysis of BTK, Aurora A, and ca-tubulin levels. Cells treated with Compound I-3 showed BTK degradation. (
Western Blotting
Cells are lysed using RIPA buffer supplemented with protease inhibitor cocktail (Roche) and phosSTOP phosphatase inhibitor cocktail (Roche) on ice for 15 minutes. The lysates are spun at 20,000×g for 15 minutes on 4° C. and protein concentration is determined using BCA assay (Pierce). The following primary antibodies are used in this study: BTK, GADPH, tubulin and Aurora A kinase (all from Cell Signaling Technology). Blots are imaged using fluorescence-labeled secondary antibodies (LI-COR) on the OdysseyCLxImager (LI-COR). Quantification of band intensities is performed using OdysseyCLx software (LI-COR).
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