Antibodies against β-actin (MAB1501R, dilution 1:500), Flag-tag (M2, dilution 1:1,000) and HA-tag (#561, dilution 1:500) were purchased from Chemicon, Sigma and MBL (Japan), respectively. The other antibodies against the proteasome subunits used in this study were described previously13 (link)43 (link) and used at a 1:1,000 dilution. Hybridoma cells (28-14-8 and Y3) were purchased from ATCC. Antibodies were purified from culture supernatants using protein G-Sepharose (GE Healthcase) and conjugated to NHS-activated Sepharose 4 Fast Flow (GE Healthcare), according to the manufacturer's protocol.
Mab1501r
Manufactured by Merck Group
Sourced in United States, United Kingdom, Japan
MAB1501R is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Irdye 800cw, by LI COR (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Quantity one 4, by Bio-Rad (2 mentions)
Pa3 16717, by Thermo Fisher Scientific (2 mentions)
Ahb0261, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Odyssey 3, by LI COR (2 mentions)
Anti mouse igg horseradish hrp linked na931v, by GE Healthcare (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Nhs activated sepharose 4 fast flow, by GE Healthcare (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Ab24525, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti mouse a4416, by Merck Group (1 mentions)
Ab34710, by Abcam (1 mentions)
56 protocols using mab1501r
1
Antibody Purification and Conjugation
2
Quantification of MDR1/P-gp Protein Levels
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The total protein contents of NLP siM and OFA siM transfected groups were extracted after 48, 72 and 96 hr. Protein extraction for control groups was carried out after 48 hr. To prepare cell lysate, the cell culture plate was placed on ice, cells were washed with 1 x PBS and mixed with P38 buffer (Tris-HCl (62.5 mM), SDS (2% W/V), glycerol (10% W/V), DTT (50 mM) in protease inhibitor pretreated nuclease-free water (half of the complete protease inhibitor cocktail tablet (Roche) dissolved in 25 ml of double distilled water). The protein concentration was determined using the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. Then cellular levels of MDR1/P-gp, in different experimental groups were analysed by western blotting as Stege et al described (15 (link)). MDR1/P-gp proteins were detected using a mouse monoclonal antibody (mAbs) C219 (Alexis, San Diego, CA, USA). We used mouse mAb against actin (mAb 1501 R; Chemicon, Temecula, CA) as a loading control and peroxidase-conjugated goat anti- mouse IgG (A-1949; Sigma, St. Louis, MO, USA) as a secondary antibody. Finally, protein bands were analyzed using UVtec software (UK). Experiments performed in triplicate.
3
Western Blot Analysis of Extracellular Matrix Proteins
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The expression of Collagen 1 a1 (Col1a1), Vimentin and alpha smooth muscle Actin α-SMA was evaluated by western blot. Cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP-40, protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor (Roche)), gently vortexed for 20 min at 4 °C and centrifuged for 15 min at 13,200 rpm at 4 °C. Supernatants were quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of proteins from cell extracts were loaded and separated in 8% SDS-PAGE. After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes (Millipore), therefore blocked (5% no fat milk in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 2% BSA; overnight at 4 °C or 2 h at room temperature): anti-α-SMA [E184] (ab32575, Abcam), anti-Vimentin (ab24525, Abcam), anti-Col1a1 (ab34710, Abcam) and anti-β-Actin (MAB1501R, Chemicon). After wash, the membranes were incubated with the appropriate HRP-conjugated secondary Ab (1:5000 in TBST + 2% BSA; 2 h at room temperature; anti-mouse A4416 and anti-rabbit A0545, Sigma). The immunoreactivity was detected by ECL reagents (Amersham), acquired with the ChemiDoc imaging system (Image Lab, Bio-Rad).
4
ApoER2 Antibody Characterization
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We used a rabbit polyclonal antibody directed against a region near the C-terminal of ApoER2 (A3481, Sigma). The rabbit polyclonal antiserum against the recombinant human ApoER2 cytoplasmic domain and the mouse monoclonal anti-HA have been described before [101 (link)]. We also used a p75NTR rabbit polyclonal antibody (07–476, Millipore), a mouse monoclonal anti-β-tubulin antibody (05–661, Millipore), a mouse monoclonal anti-actin antibody (MAB1501R, Chemicon), a rabbit polyclonal anti-AKT antibody (#9272, Cell Signaling), a rabbit monoclonal anti-phosphorylated AKT antibody (#4060, Cell Signaling), a mouse monoclonal anti-phosphorylated ERK antibody (sc-7383, Santa Cruz Biotechnology), a rabbit polyclonal anti-Dab1 antibody (AB5840, Chemicon International), and a mouse monoclonal anti-MAP2 antibody (MAB378, Chemicon). We used horseradish peroxidase (HRP)-conjugated secondary antibodies (Chemicon) and Alexa 555- and 488-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Molecular Probes).
5
Neuroproteomics: Antibody Staining Protocols
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Primary antibodies against CAMK2A (Novus Biologicals NB100‐1983, 1:500 for immunohistochemistry (IHC)), Parvalbumin (SWANT, 1:9000 for IHC), NeuN ( 1:1000 IHC), TSC1 (CST #4906, 1:1000 for western blot), RHEB1 (CST #4935, 1:1000 for western blot), pS6S240/244 (CST #5634, 1:1000 for IHC, 1:2000 for western blot), S6 (CST #2217, 1:2000 for western blot), pAktSer473 (CST #9271 1:2000 for western blot) and Actin (Chemicon MAB1501R, 1:20.000 for western blot) were used.
6
Western Blot Analysis of UBE3A in Mouse Brain
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Mice were euthanized by cervical dislocation. The cortex, hippocampus, striatum, and cerebellum were extracted. Brain sections were homogenized and lysed in 2× Laemmli buffer, with 1% protease inhibitor, by sonicating 3 times for 3 seconds. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, 23225). Twenty micrograms of proteins were separated on a precast 4–12% Criterion XT Bis-Tris gel (Bio-Rad) and transferred to a nitrocellulose membrane using TurboBlot (Bio-Rad). The following antibodies were used: anti-UBE3A (anti-E6AP, MilliporeSigma, catalog E8655, 1:1000) and anti-actin (Chemicon, MAB1501R, 1:20,000). The next day, the membranes were probed with secondary goat anti-mouse antibody (LI-COR Biosciences, IRDye 800CW, 926-32210, 1:15,000). The membranes were scanned using Odyssey CLx (LI-COR Biosciences) and quantified using the Odyssey 3.0 software.
7
MDCK Cell Protein Analysis
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Whole cell lysates of MDCK cells were prepared by homogenization in RIPA buffer. The whole cell lysate of MDCK cells was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 5-LOX and actin were analyzed by immunoblotting using an anti-5-LOX monoclonal antibody (ab169755, dilution 1/1,000; Abcam, Cambridge, UK) and anti-actin antibody (MAB1501R, dilution 1/2,000; Chemicon International, Temecula, CA).
8
Brain CAMK2 Protein Expression Analysis
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Mice were anaesthetized using isoflurane and sacrificed by decapitation. Brain samples were taken out quickly and stored in liquid nitrogen. Upon protein determination lysates were first prepared and brain samples were homogenized in lysis buffer (10 mM Tris-HCl 6.8, 2.5% SDS, 2 mM EDTA). Protein concentration in the samples was determined and then lysate concentrations were adjusted to 1 mg/ml. Western blots were probed with primary antibodies against either CAMK2A (6G9, 1:40.000, Abcam), CAMK2B (CB-β1, 1:10.000, Invitrogen), Actin (MAB1501R, 1:20.000, Chemicon) or Ph-T286/T287 (autophosphorylated α CAMK2A and CAMK2B antibody; #06–881; 1:5000; Upstate Cell Signaling Solutions) and secondary antibodies (goat anti-mouse and/or goat anti-rabbit, both 1:3000, AffiniPure #115-007-003 and #111-007-003). Blots were stained with Enhanced ChemoLuminescence (ECL) (#32106, Pierce) or stained and quantified using LI-COR Odyssey Scanner and Odyssey 3.0 software. Quantification of western blot in ECL was done using ImageJ.
9
Schwann Cell Reelin Signaling Activation
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Cells lysates were prepared using lysis buffer (50 mM Tris, pH 7.4, 2 mM EDTA, 1% PMSF, 1% Triton X-100 and protease inhibitors). The proteins in the cell lysates were denatured and then separated on SDS-PAGE gels. The electrophoretically separated proteins were transferred to nitrocellulose membranes, blocked with 5% no-fat milk, and immunoblotted with the respective antibodies. The bound antibodies were detected by using the enhanced chemiluminescence (ECL) system (Amersham Biosciences). To determine activation of Reelin signaling in Schwann cells the antibodies were mouse monoclonal anti-actin (MAB1501R, Chemicon), rabbit polyclonal anti-AKT antibody (#9272, Cell Signaling) and rabbit monoclonal anti-phosphorylated AKT antibody (#4060, Cell Signaling).
10
Culturing and Analyzing Postnatal Hippocampal Neurons
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