An ASMase assay kit (ab190554, Abcam) was used to assay ASMase activity in HUVECs. Briefly, the cells were lysed with mammalian cell lysis buffer (ab179835, Abcam) and then the samples were reacted with ASMase assay reagents according to the protocol recommended by the manufacturer. After the incubation, fluorescence from each sample was detected at Ex/Em=540/590 nm using a microplate reader.
Ab179835
Manufactured by Abcam
Sourced in United States
Ab179835 is a primary antibody that recognizes the protein target XYZ. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab190554, by Abcam (1 mentions)
Ab219926, by Abcam (1 mentions)
Beadblaster24r, by Benchmark Scientific (1 mentions)
Ecl western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Protran, by Cytiva (1 mentions)
Ab138881, by Abcam (1 mentions)
Ab204708, by Abcam (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
6 protocols using ab179835
1
Quantifying Acid Sphingomyelinase Activity
2
Glutathione Peroxidase Activity Assay
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Snap-frozen lung tissues (10–15 mg) were homogenized in 300 µL 1× Mammalian Cell Lysis Buffer (ab179835, Abcam) in a BeadBlaster24R (Benchmark) using a linear speed of 4 m/s for two 45 s cycles. Gpx activity (ab219926, Abcam) measurement was based on a series of reactions that ended with a NADP signal that reacted with NADP+ to give a fluorescent signal (420 nm for excitement/480 nm for emission) that was directly proportional to GPx activity.
3
Western Blot Analysis of PRKAR2B
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Cells were harvested and extracted using 1× mammalian lysis buffer (ab179835; Abcam) supplemented with protease and phosphatase inhibitor cocktail (78440; Thermo Fisher). The protein concentration was determined using the Bradford protein assay. Cell lysates were subjected to Western blot analysis using conventional SDS/PAGE and protein transfer to nitrocellulose filters (Protran, Whatman). The membrane was blocked using 5% nonfat milk-TBST (Tris-buffered saline with Tween 20) (for anti-PRKAR2B and anti-GAPDH), or 3% nonfat milk-PBST (phosphate-buffered saline with Tween 20) (for anti-actin antibody) overnight at 4°C. The antibodies used for immunoblotting were as follows: rabbit polyclonal antibody anti-GAPDH (ABS16; Merck Millipore), mouse monoclonal antibody anti-PRKAR2B (610625; BD Transduction Laboratories), and goat polyclonal antibody anti-actin (Santa Cruz Biotechnologies; I-19). After washing, proteins were visualized using enhanced chemiluminescence (ECL) Western blotting detection reagents (Thermo Scientific) with a fusion instrument. The beta-actin was used as a loading control throughout all experiments.
4
Determining Cellular Glutathione Levels
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BMDMs were established as described and 5 × 106 of BMDMs were pelleted by centrifugation, the pellet was lysed in mammalian lysis buffer (Abcam, ab179835), incubated 10 min at RT and centrifuged at top speed at 4°C 15 min. Supernatant was transferred to a fresh tube and used for deproteinization following manufacturer's instructions (Abcam, ab204708). The resultant supernatant was used for determining reduced GSH, total GSH and oxidized GSSG was calculated as per manufacturer’s instructions (Abcam, ab138881).
5
Intracellular Calcium Measurement in Cells
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Cells were seeded in black 96-well plates with black bottoms at a density of 20,000 cells/well and left to adhere overnight. The following day, intracellular calcium was measured using the Cal-520 AM kit (Abcam, Cambridge, UK, ab171868). Cells were washed with HHBS (AAT Bioquest, Pleasanton, CA, USA, 20011) and incubated with 10 µM Cal-520 in HHBS supplemented with 10% FBS for 90 min at 37 °C, followed by 30 min at room temperature. Afterward, half of the wells were treated for 3 min with 100 µM ATP (Sigma, A6419) in HHBS, while the other half were incubated with HHBS only. Fluorescence was measured using the microplate reader with the following parameters: excitation/emission = 490/525 nm.
Afterward, cells were washed with PBS (Life Technologies, 14190094) and mechanically lysed using 1× mammalian lysis buffer (Abcam, ab179835) freshly supplemented with protease inhibitors (Merck, Rahway, NJ, USA, 535140) by pipetting up and down and scraping the bottom of the wells with the pipette tips. After 15 min of incubation, cells were centrifuged at 435× g for 5 min, and the supernatant was transferred to a clean plate for protein quantification.
Afterward, cells were washed with PBS (Life Technologies, 14190094) and mechanically lysed using 1× mammalian lysis buffer (Abcam, ab179835) freshly supplemented with protease inhibitors (Merck, Rahway, NJ, USA, 535140) by pipetting up and down and scraping the bottom of the wells with the pipette tips. After 15 min of incubation, cells were centrifuged at 435× g for 5 min, and the supernatant was transferred to a clean plate for protein quantification.
6
Protein Quantification in MDA-MB231 Cells
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The MDA-MB231 cells were plated (3 × 106) in two 10 cm plates and
grown in treated media and control media. Media were changed daily until
confluence. BCA Protein Assay Reagent Kit (Pierce 23225 23227) is a
detergent-compatible formulation based on BCA for the colorimetric detection of
quantitation of total protein was used for this analysis. This method utilizes a
well-known reduction of copper by protein in an alkaline medium with sensitive
and selective colorimetric detection of the cuprous cation. The cells were
trypsinized and pelleted using 500 r/min for 5 min, then lysed in 300 µl of
mammalian cell lysis buffer (Abcam ab179835). Total protein concentration was
determined using the microplate procedure and the dilution parameters of 0, 200,
400, 600, 800, and 100 µg/ml were made according to the preparation of diluted
albumin (BSA) standards with 50 parts of BCA Reagent A with 1 part BCA Reagent B
(50:1, Reagent A: B). Once the dilutions were made, 25 µl of each standard and
unknown sample replicates were pipetted into the microplate wells. Prior to
placing wells in a 37°C incubator for 30 min, 200 µl of the working reagent was
added to each well. The plate was then cooled to room temperature and measured
at the absorbance of 562 nm on a plate reader.
grown in treated media and control media. Media were changed daily until
confluence. BCA Protein Assay Reagent Kit (Pierce 23225 23227) is a
detergent-compatible formulation based on BCA for the colorimetric detection of
quantitation of total protein was used for this analysis. This method utilizes a
well-known reduction of copper by protein in an alkaline medium with sensitive
and selective colorimetric detection of the cuprous cation. The cells were
trypsinized and pelleted using 500 r/min for 5 min, then lysed in 300 µl of
mammalian cell lysis buffer (Abcam ab179835). Total protein concentration was
determined using the microplate procedure and the dilution parameters of 0, 200,
400, 600, 800, and 100 µg/ml were made according to the preparation of diluted
albumin (BSA) standards with 50 parts of BCA Reagent A with 1 part BCA Reagent B
(50:1, Reagent A: B). Once the dilutions were made, 25 µl of each standard and
unknown sample replicates were pipetted into the microplate wells. Prior to
placing wells in a 37°C incubator for 30 min, 200 µl of the working reagent was
added to each well. The plate was then cooled to room temperature and measured
at the absorbance of 562 nm on a plate reader.
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