Tissues and cells were lysed in cold radioimmunoprecipitation assay lysis buffer. The proteins were separated with 10% SDS-PAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated overnight with phosphate-buffered saline containing 5% milk at 4°C. Following incubation, the PVDF membrane was incubated with monoclonal mouse anti-human Spry2 (1:200; ab60719) and monoclonal mouse anti-human GAPDH primary antibodies (1:200; ab125247; Abcam, Cambridge, UK) at room temperature for 3 h, and then incubated with polyclonal rabbit anti-mouse secondary antibodies (1:5,000; ab175743; Abcam) at room temperature for 1 h. An electrochemiluminescence kit (Pierce Chemical, Rockford, IL, USA) was then used to perform chemiluminescent detection. The relative protein expression was analyzed by Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and was presented as the density ratio versus GAPDH.
Ab125247
Manufactured by Abcam
Sourced in United States
Ab125247 is a lab equipment product from Abcam. It is a microscope slide that can be used for various microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ab231174, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab191866, by Abcam (2 mentions)
Ab6728, by Abcam (2 mentions)
Ab6160, by Abcam (2 mentions)
Ab75972, by Abcam (2 mentions)
Ab175186, by Abcam (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab6789, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Bca kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Image pro plus software 6, by Media Cybernetics (1 mentions)
Anti hybid antibody, by Proteintech (1 mentions)
Envision system hrp labeled polymer anti rabbit igg, by Agilent Technologies (1 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Ma3 926, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
32 protocols using ab125247
1
Western Blot Analysis of Spry2 Protein
2
Western Blot Analysis of OA Chondrocytes
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Cell lysates of OA chondrocytes, which were treated with the cytokines or siRNAs for HYBID or TMEM2 (see below), were harvested with 2× sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 2-mercaptoethanol. The proteins resolved on the gels by SDS-PAGE were transferred onto PVDF membranes, and they were reacted with anti-HYBID antibody (Proteintech, Rosemont, IL), anti-TMEM2 antibody (SAB2105088; Sigma-Aldrich) and anti-GAPDH antibody (as a loading control) (ab125247, Abcam), followed by incubation with Envision + System HRP-labeled polymer anti-rabbit IgG (Dako, Glostrup, Denmark)12 (link). The intensity of the immunoreactive bands detected by chemiluminescence imaging system using Amersham Imager 680 (GE Healthcare Life Sciences, Tokyo, Japan) was quantified by densitometric analysis using ImageJ software (http//rsb.info.nih.gov/ij/).
3
Molecular Profiling of Cardiac Calcium Handling Proteins
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Homogenates were prepared from the left atrial or cell samples and lysed using mixed cell lysates (RIPA: PMSF: phosphatase inhibitor cocktail = 100: 1: 1, Solarbio, China). Protein concentration was measured using a BCA assay kit (PC0020, Solarbio, China). Mini-PROTEAN Tetra (Bio-rad) electrophoresis was performed with 20 μg of protein loaded onto a 10% SDS-PAGE gel (80 V, 140 min). Proteins were transferred on PVDF membranes by wet transfer (300 mA, 90 min). The blocking process was performed in 5% BSA blocking solution at room temperature for 1 h, while the PVDF membranes were incubated with the primary antibodies overnight at 4°C. The following primary antibodies were utilized: anti-CaMKII (PA5-22168, Thermo Fisher, U.S.A.); anti-phospho-CaMKII (LS-C354565, Lifespan, U.S.A.); anti-RyR2 (MA3-916, Thermo Fisher, U.S.A.); anti-phospho-RyR2 (LS-C358303, Lifespan, U.S.A.); anti-SERCA (MA3-910, Thermo Fisher, U.S.A.); anti-PLB (MA3-922, Thermo Fisher, U.S.A.); anti-phospho-PLB (ab15000-50, Abcam, U.S.A.); NCX (MA3-926, Thermo Fisher, U.S.A.); and GAPDH (ab125247, Abcam, USA).
4
Microglial Protein Expression Analysis
5
Protein Extraction and Quantification Protocol
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The total protein in the cells and tissues was extracted using radioimmunoprecipitation assay (RIPA) buffer (Auragene, Changsha, China). The proteins were quantified using a BCA protein assay kit (Thermo Scientific). The protein samples were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by transfer of the proteins to polyvinylidene fluoride membranes and blocking of the membranes using 5% Tris buffered saline with Tween-20 buffer containing 5% skim milk. Anti-MAPRE2 (1:100, ab45767; Abcam, Cambridge Science Park, UK), anti-WDHD1 (1:100, ab72436; Abcam), anti-DSTN (1:500, ab186754; Abcam), and anti-ubiquitin antibodies (1:200, 10201-2-AP; Proteintech, Wuhan, China) were used as primary antibodies and incubated with the membrane overnight at 4°C. The membrane was then washed and incubated with a secondary horseradish peroxidase–labeled goat anti–rabbit immunoglobulin G (IgG) antibody (1:4,000; Abcam) for 1 h at room temperature. Immunoassay was performed by enhanced chemiluminescence detection system (ECL; Cell Signaling Technology, Danvers, MA, USA) combined with Western blot system (Auragene). Strip signal strength was checked using the software IPP6.0. GAPDH (1:4,000, ab125247; Abcam) was used as an internal control to normalize the expression of other proteins.
6
Western Blot Analysis of Protein Samples
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Proteins were denatured 3 min at 95°C, separated on 4–12% Bis‐Tris Gel (NuPAGE), transferred on nitrocellulose membranes (Whatman Protran), blocked with 5% non‐fat dry milk in Tris‐buffered saline (TBS) buffer, incubated with anti‐GFP (ab290, Abcam), HA (ab130275, Abcam), C9ORF72 (3H10, homemade), and GAPDH (ab125247, Abcam) in TBS plus 5% non‐fat dry milk, washed three times, and incubated with anti‐rabbit or mouse peroxidase antibody (1:10,000, Cell Signaling) 1 h in TBS, followed by washing and ECL chemiluminescence revelation (Amersham ECL Prime).
7
Quantifying Recombinant ADAMTS13 Expression
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HeLa and HEK293 cells were obtained from ATCC (Manassas, VA); and human hepatic stellate cells from Creative Bioarray (Shirley, NY). Monolayer cell culture was prepared per supplier instructions for mRNA transfection. Briefly, cells with 80% confluency were transfected with 2.5 µg of each mRNA and 4 uL Lipofectamine™ MessengerMAX™ transfection reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Cell lysates and culture media were collected at the indicated time points for capillary gel electrophoresis or SDS-PAGE followed by immunoblotting with anti-hADAMTS13 (rabbit mAb ab177940, 1:2,000) or anti-GAPDH (mouse mAb ab125247, 1:6,000, both from Abcam, Cambridge, MA) and detection with the corresponding secondary antibodies. The collected culture media were also subjected to quantitative measurements of rhADAMTS13 as below.
8
Quantitative Western Blot Analysis
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MDA-MB-435 and MDA-MB-231 cells were lysed with lysis buffer (Beyotime, China). The extract samples were separated by 10% SDS-PAGE gel and then transferred to NC membrane for a conventional Western blotting analysis. The membranes were probed with primary antibodies to CDC27 (1:3000, ab72214, Abcam) and GAPDH (1:2000, ab125247, Abcam) and then incubated with corresponding HRP-conjugated secondary antibody (1:10000, anti-rabbit IgG (HRP) ab191866, Abcam; 1:10000, anti-mouse IgG (HRP), ab6728, Abcam). The signals were visualized by using ECL Western Blotting Substrate (Thermo Scientific Pierce) and the signal intensity was quantified by using Image-J software.
9
Evaluating Protein Expression in Transfected COS7 Cells
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After transfection with double-gene plasmids, COS7 cells were cultured at 37 °C for 4 h, and then changed to fresh DMEM containing 10% FBS. A plasmid-free negative control group was established. Cells were collected for test 48 h after transfection. The cells were mixed within ice-precooled protein lysates (l mM PMSF, pH7.4), broken up by ultrasonic cracker, centrifuged at 4 °C, 12000 rpm for 15 min, and the supernatant was taken for loading sample preparation and Western blotting assay. Antibodies used: anti-4-1BBL mouse monoclonal antibody (sc-398933, Santa cruz, 1:1000), anti-CEACAM6 rabbit monoclonal antibody (ab235139, Abcam, 1:1000), anti-PD-L1 rabbit polyclonal antibody (ab233482, Abcam, 1:1000), and anti-GAPDH mouse monoclonal antibody (ab125247, Abcam, 1:5000), and secondary antibodies (Beyotime Biotechnology). The follow-up WB method was as reported [36 (link)].
10
Immunoblotting of GFP in H1299 cells
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Western blot was performed following the routine protocol. H1299 cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, pH 8.0, 30 mM NaF, 1 mM Na3VO4, 40 mM β-glycerophosphate, 0.1 mM PMSF, 10% glycerol and 1% Nonidet-P40) and equal total protein of cell lysates from each condition were resolved by 15% SDS-PAGE followed by immunoblotting. The primary antibodies were as the following: anti-GFP (ab183734, Abcam, MA, USA), 1:10,000; anti-GAPDH (ab125247, Abcam, MA, USA).
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