Experimental cells were harvested and then mixed with RIPA:PMSF (100:1) to facilitate total protein extraction, and protein concentration was then determined using a bicinchoninic acid kit (BOSTER). Equal concentrations (40 μg) of total protein were then separated by electrophoresis on a 10% separation gel and transferred to a polyvinylidene fluoride membrane. This membrane was then blocked for 1 h using nonfat milk (5%) and incubated overnight with a working solution of the relevant primary antibodies (EFEMP2 12004-1-AP, STEAP1 20199-1-AP, STEAP2 20201-1-AP, Twist 25465-1-AP, Proteintech; STEAP3 PA5-115969, STEAP4 PA5-115971, Invitrogen™; EMT Antibody Sampler Kit #9782, Cell Signaling Technology; PI3K ab86714, p-PI3K ab182651, AKT ab8805, p-AKT 38449, mTOR ab32028, p-mTOR ab109268, Abcam) at 4°C. The membranes were then washed using Tris-buffered saline with Tween® 20 and incubated with the secondary antibody for 1 h at room temperature. Membranes were then washed again and visualized using an enhanced chemiluminescent substrate kit (Millipore) and Image J software.
Enhanced chemiluminescent substrate kit
Manufactured by Merck Group
Sourced in United Kingdom
The Enhanced Chemiluminescent Substrate Kit is a laboratory reagent designed to facilitate the detection and quantification of proteins in Western blot analysis. The kit provides a chemiluminescent substrate that reacts with the enzyme-labeled secondary antibody, producing a light signal that can be captured and measured using a compatible detection system.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid kit, by Boster Bio (1 mentions)
Efemp2 12004 1 ap, by Proteintech (1 mentions)
Twist 25465 1 ap, by Proteintech (1 mentions)
Emt antibody sampler kit 9782, by Cell Signaling Technology (1 mentions)
P mtor ab109268, by Abcam (1 mentions)
Pparγ, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ab2051, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab2302, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Runx2, by Abcam (1 mentions)
Collagen 1 col 1, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
6 protocols using enhanced chemiluminescent substrate kit
1
Protein Expression Analysis of EMT Markers
2
Western Blot Analysis of Osteogenic Markers
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GMSCs were seeded in 6-well plates at 105 cells/well. GMSCs were washed with PBS, lysed with RIPA lysis buffer containing 1% PMSF (Solarbio) and then centrifuged at 12,000 ×g for 15 mins at 4°C. After centrifugation, proteins in a supernatant were collected and quantified using a BCA protein assay kit (Solarbio). Total proteins were mixed with 5X sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and incubated at 100°C for 5 mins. Twenty micrograms of protein per lane was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked and probed with primary antibodies that recognized ALP and Osterix (1:10,000; Abcam, Cambridge, MA, USA) and β-catenin, GSK3β, p-GSK3β (Ser9), LEF, PPARγ and GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were selected according to the species of origin of the primary antibodies. Blotted proteins were detected using an enhanced chemiluminescent substrate kit (Millipore). The level of each protein was normalized to that of GAPDH before statistical analysis. ImageJ software (NIH, Bethesda) was used to quantify protein expression.
3
Quantitative Western Blot Analysis
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Western blot analysis was performed as previously described [23 (link)]. Briefly, protein was extracted and loaded on a 10% polyacrylamide gel. Then, proteins were transferred onto Polyvinylidene Fluoride (PVDF; Millipore, Bedford, MA, USA), and incubated with primary antibody including ADAM17 (ab2051, 1:1000, Abcam, Cambridge, MA, USA), Bcl-2 (ab182858, 1:2000, Abcam), Cleaved caspase-3 (ab2302, 1:1000, Abcam), E-cadherin (14472, 1:1000, Cell signaling technology, Boston, MA, USA), Vimentin (5741, 1:1000, 5741, Cell signaling technology) and GAPDH (ab8245, 1:6000, Abcam) overnight at 4 °C. GAPDH was used as a internal control. Then, the membranes were incubated with secondary antibody (Abcam) for 40 min at temperature, and an enhanced chemiluminescent substrate kit (Millipore) was employed to visualize the unique proteins.
4
Western Blot Analysis of Cellular Markers
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The protocol of this Western blot was in the light of the previous description [24 (link)]. Briefly, the protein extracts with the sample buffer were denatured for 10 min in boiling water, followed by dissociated on a 10% polyacrylamide gel. The isolated protein were transfected onto the Polyvinylidene Fluoride (PVDF; Millipore, Bedford, MA, U.S.A.), and then maintained with primary antibodies (Abcam, Cambridge, MA, U.S.A.), including CD14 (ab183322, 1:1000), CD44 (ab189524, 1:1000), CD90 (ab225, 1:1000), ADAMTS9 (ab32565, 1:2000), LC3B (ab51520, 1:3000), p62 (ab109012, 1:25000), Cleaved-caspase-3 (active large subunit; ab13847, 1:500), BCL2-Associated X (Bax; ab32503, 1:7000), B-Cell leukemia/lymphoma 2 (Bcl-2; ab32124, 1:1000), β-actin (ab8226, 1:6000), and GAPDH (ab8245, 1:5000), of which β-actin and GAPDH acted as the references. Then. the membranes were incubated with corresponding secondary antibody (Abcam). The bands were visualized using an enhanced chemiluminescent substrate kit (Millipore).
5
Evaluating Osteogenic Potential of Puerarin
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After culturing in osteogenic inducing medium containing different concentrations of puerarin (0 and 10−5 mol/L) for 14 days, BMSCs were lysed with RIPA lysis buffer containing 1% PMSF (Solarbio, Beijing, China). The total collected protein concentrations were quantified by a BCA protein assay kit. All protein samples (20μg) were denatured and separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto 0.45-μm polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). Afterwards, the membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with primary antibodies that recognized β-catenin (1:800; Cell Signaling Technology, Danvers, MA, USA), GAPDH, ALP, Runx2, Collagen I (Col I) (1:1000; Abcam, Cambridge, MA, USA) overnight at 4 ℃. After washing in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with a secondary antibody (Absin, Shanghai, China) solution at 37 ℃ for 1 h. Secondary antibodies were selected based on the source of primary antibodies. An enhanced chemiluminescent substrate kit (Millipore) and a chemical imaging system (Amersham Imager 600; GE Healthcare, Little Chalfont, UK) were used to detect immunoreactive proteins. GAPDH was used as internal reference.
6
Protein Extraction and Immunoblotting in BT474/OX and MCF-7/OX Cells
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Extraction of total protein from BT474/OX or MCF-7/OX cells was conducted with RIPA buffer (Sigma-Aldrich). Then, total protein quantitation was performed by Bradford method. Then, protein samples were subjected to SDS-PAGE and transferred onto PVDF membranes (Roche, Switzerland). After blocked with 5% skim milk, the PVDF membranes were cultivated with primary antibodies against MRP1 (1:1000; ab260038; Abcam), MDR1 (1:1000; #13,978; Cell signaling technology), LRP1 (1:1000; ab92544; Abcam), Notch2 (1:1000; ab245325; Abcam), GSK-3β (1:1000; #12,456; Cell signaling technology), β-catenin (1:1000; ab68183; Abcam), or β-actin (1:1000; ab8227; Abcam) at 4°C overnight and incubated with secondary antibody for 2 hours at room temperature. The protein bands were visualized with an enhanced chemiluminescent substrate kit (Millipore). The proteins were quantified using Quantity One software (Bio-Rad Laboratories, USA).
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