Antibodies used were as follows: rabbit anti-human LC3 antibody (NB100-2220, Novus Biologicals, Madrid, Spain), mouse anti-human LAMP-1 antibody [H4A3] (ab 25630, Abcam, Cambridge, United Kingdom), mouse anti-human β-Glucocerebrosidase antibody (GBA) (B-6) (sc-166407, Santa Cruz, Heidelberg, Germany), rabbit recombinant anti-rab10 (phospho T73) antibody (ab230261, Abcam), rabbit monoclonal anti-Rab10 (8127S, Cell Signaling, Danvers, MA, United States), mouse anti-human β-Actin antibody (A1978-200UL, Sigma, Merck Life Science, Madrid, Spain), anti-rabbit antibody (A0545, Sigma, Merck Life Science, Madrid, Spain) and anti-mouse antibody (A3682, Sigma).
A3682
Manufactured by Merck Group
Sourced in Spain
A3682 is a laboratory equipment product. It is used for performing various scientific experiments and analysis in a laboratory setting. The core function of this product is to provide reliable and consistent results for research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab230261, by Abcam (1 mentions)
Nb100 2220, by Novus Biologicals (1 mentions)
Ab25630, by Abcam (1 mentions)
West pico chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
H1029, by Merck Group (1 mentions)
Las4000, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Calsequestrin, by Dianova (1 mentions)
Pp 1α, by Santa Cruz Biotechnology (1 mentions)
Pp2ac, by Merck Group (1 mentions)
A0545, by Merck Group (1 mentions)
Mouse anti cd63, by Thermo Fisher Scientific (1 mentions)
Mouse anti cd81, by Thermo Fisher Scientific (1 mentions)
Rabbit anti grp94, by Abcam (1 mentions)
Mouse anti gm130, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Mouse anti gapdh, by HyTest (1 mentions)
Dasatinib, by Merck Group (1 mentions)
Imatinib mesylate, by Merck Group (1 mentions)
6 protocols using a3682
1
Antibody Characterization for Lysosomal Disorders
2
Liposome Association Assay for His-Tagged Protein
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The liposome association assay was performed as described25 (link)26 (link). Lipids were dried and incubated in Tris buffered saline (TBS) (50 mM Tris-HCl, pH 7.0, 0.1 M NaCl) at 37 °C for 1 h. The liposomes were formed by vigorous vortexing (5 min), pelleted by centrifugation at 20,000 × g for 10 min at 4 °C, and washed twice with ice-cold TBS. Purified His-FT was added to the liposomes to a final 100 μl volume, and the mixture was incubated at 30 °C for 30 min. After incubation, liposomes were pelleted again by centrifugation at 20,000 × g and washed twice with ice-cold TBS. The pellets were analysed by SDS–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane (Bio-Rad, Hercules, CA) for protein gel blot analysis. His-FT was immunodetected by a monoclonal antibody against penta-His (H1029, Sigma-Aldrich; dilution 1:1,000) and horseradish peroxidase–coupled anti-mouse IgG (A3682, Sigma-Aldrich; dilution 1:10,000) with the West Pico chemiluminescence detection kit (Thermo Scientific, Rockford, IL) and signal intensity of each spot was quantified by image analyser (LAS4000, GE healthcare). An uncropped scan of a representative result is shown in Supplementary Fig. 6 .
3
Cardiac Protein Phosphorylation Analysis
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After incubation with H2O2, NRCMs were lysed with ice-cold GST-fish buffer (10% glycerol, 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-4, 4 mM MgCl2, 1% IGEPAL CA-630, complete protease inhibitor), cell debris was removed at 13,000 g (10 min, 4 °C). For non-reducing 12–15% acrylamide SDS-PAGE, the cells were lysed in the presence of 10 mM maleimide but without DTT and heat. Gels were blotted onto a nitrocellulose membrane, blocked with 1x Roti-Block (Roth) for 1 h, washed with TBST (Tris-buffered saline, 0.1% Tween 20) and incubated overnight at 4 °C with a primary antibody: calsequestrin (1:1000, Source: rabbit, Dianova, ABR-01164), PP-1α (1:200, Source: goat, Santa-Cruz, sc-6104), PP-2Ac (1:1000, Source: rabbit, Millipore, 07–324), PKA-RI (1:200, Source: mouse, BD Transduction laboratories, 610166), PLB-pSer16 (1:5000, Source: rabbit, Badrilla Ltd., A010–12), cMyBPC-pSer282 (1:5000, Source: rabbit, Enzo Life Science, ALX-215–057-R050), and I-1-pThr35 (1:1000, Source: rabbit, Cell signalling, #2302). The membrane was washed three times with TBST for 10 min each and incubated for 1 h with the secondary antibody: HRP-coupled antibodies (mouse: Sigma-Aldrich, A3682; rabbit: Sigma-Aldrich, A0545; goat: Santa-Cruz, sc-2020).
4
Exosomal Protein Extraction and Analysis
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Total protein was extracted from exosomes with procedures as described in detail elsewhere [64 (link)]. Briefly, equal amounts of proteins were added on 4–12% SDS gels for electrophoresis and then transferred to PVDF membranes (ThermoFisher Scientific). The membranes were blocked for 1 h in 5% milk. All the membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies used in this study were as follows: rabbit-anti-CD9 (Thermo Fisher Scientific, PA5-11559), mouse-anti-CD63 (Thermo Fisher Scientific, 10628D), mouse-anti-CD81 (Thermo Fisher Scientific, MA5-13548), rabbit-anti-GRP94 (Abcam, 13509), mouse-anti-GM130 (Santa Cruz, sc-55591), mouse-anti-GAPDH (HyTest, 5G4MAb6C5). On the next day, the membranes were washed three times for 10 min in TBST (pH = 7.4) and incubated for 1 h with secondary antibodies at room temperature. Secondary antibodies used were anti-Mouse IgG (Fab specific)-Peroxidase antibody produced in goat (Sigma-Aldrich, A3682) and anti-Rabbit IgG (whole molecule)-Peroxidase antibody produced in goat (Sigma-Aldrich, A0545). The results were visualized with chemiluminescence, and the density of the bands was analyzed by ImageJ software.
5
Chronic myeloid leukemia cell line K-562 inhibition
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Chronic myeloid leukaemia cell line K‐562 was cultured in RPMI‐1640 media (Invitrogen, Thermo Scientific) with 10% FBS (HiMedia) at 37°C in 5% CO2. Imatinib mesylate (Sigma–Aldrich; SML1027), dasatinib (Sigma–Aldrich, SML2589), and bosutinib (Bonitar 100 mg) were used as inhibitors for BCR‐ABL1 protein. PD184352 (Sigma–Aldrich; PZ0181), A‐395 (Adooq Biosciences; A12999), and IPA‐3 (Sigma–Aldrich; I2285) were used as inhibitors for MEK/ERK kinase, PRC2 complex, and PAK1 kinase respectively. Antibodies used were FUBP3 (sc‐398,466; E8), GAPDH (CST‐14C10), Histone H3 (CST‐D1H2), beta‐tubulin (Sigma–Aldrich N6786), p44/42 MAPK (ERK1/2) (CST‐137F5), phospho‐p44/42 MAPK (ERK1/2) (CST‐D13.14.4E), p38 MAPK (CST‐D13E1), phospho‐p38 MAPK (CST‐D3F9), SAPK/JNK (CST‐9252), phospho‐SAPK/JNK (CST‐81E11), SUZ12 (CST‐D39F6), EZH2 (CST‐D2C9), EED (CST‐E4L6E), RBBP4/7 (RBAP48/RBAP46) (CST‐4633), Mouse IgG (CST‐G3A1), Mouse HRP (Sigma–Aldrich A3682) and Rabbit HRP (Sigma–Aldrich AQ132P).
6
Immunoblotting Protocol for Protein Analysis
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For immunoblotting, RIPA buffer (150 mM NaCl, 50 mM Tris HCl, pH 8, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail [Roche]) was added to cell fractions or whole cells in lysis buffer for 30 min on ice and pelleted. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated in NuPAGE 4%–12% Bis-Tris mini protein gels (Thermo Fisher Scientific) and electro-transferred onto PVDF membranes using iBlot 2 (Thermo Fisher Scientific). Membranes were blocked with 5% milk in TBS with Tween-20 for 2 h and incubated overnight at 4°C with primary antibodies—anti-ELAVL1 (3A2, sc-5261, Santa Cruz Biotechnology), anti-GAPDH (G8795, Sigma-Aldrich), anti-Lamin A + Lamin C (EPR4100, Abcam), anti-PATZ1 (sc390577, Santa Cruz Biotechnology), and anti-MAP3K5 (SAB4300398, Sigma-Aldrich). Membranes were washed and incubated with horseradish peroxidase-conjugated goat antimouse or antirabbit antibodies (A3682 and A9169, Sigma-Aldrich). After washing, proteins were detected with ECL reagent (Bio-Rad), according to the manufacturer's protocol.
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