Proteins separated by nonreducing or reducing SDS–PAGE were blotted onto a PVDF membrane (Millipore), blocked with 2% Tween‐20, and equilibrated in 50 mM Tris–HCl, 500 mM NaCl, 0.1% Tween‐20, pH 9.0 (TST). The blots were incubated overnight at room temperature with an antiserum raised against the N‐terminal laminin G‐like module of PAPP‐A2 diluted 1:10,000 in TST containing 0.5% fetal bovine serum (Winn et al, 2009 ). The blots were incubated for 1 h at room temperature with polyclonal swine anti‐rabbit IgG‐HRP (DAKO, P0217) diluted 1:2,000 in TST containing 0.5% fetal bovine serum. All washing between the steps was done with TST. The blots were developed using enhanced chemiluminescence (ECL Prime, GE Healthcare), and images were captured and analyzed using an ImageQuant LAS 4000 instrument (GE Healthcare). For quantification of mutant PAPP‐A2 (A1033V), a standard curve was generated based on serial dilutions of wild‐type PAPP‐A2 of known concentration.
Imagequant las 4000 instrument
Manufactured by GE Healthcare
Sourced in United States
The ImageQuant LAS 4000 instrument is a chemiluminescence and fluorescence imaging system designed for the detection and analysis of proteins, nucleic acids, and other biological molecules separated by gel electrophoresis or spotted on membranes. The system utilizes a high-resolution CCD camera and specialized optics to capture and analyze images of labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (22 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ecl prime, by GE Healthcare (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Amersham ecl chemiluminescent reagent, by GE Healthcare (3 mentions)
Amersham ecl, by Cytiva (3 mentions)
Ecl prime western blotting detection system, by GE Healthcare (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (2 mentions)
Protease inhibitor and phosphatase inhibitor, by Roche (2 mentions)
Polyclonal swine anti rabbit igg hrp, by Agilent Technologies (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Pro prep protein extraction solution kit, by iNtRON Biotechnology (1 mentions)
Goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
27 protocols using imagequant las 4000 instrument
1
Quantification of Mutant PAPP-A2 by Western Blot
2
KPNA2 Expression in Cholangiocarcinoma
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Western blot analysis was used to confirm KPNA2 protein expression in cholangiocarcinoma cell lines. Total proteins, nucleoproteins, and cytoplasmic proteins were extracted from TFK1 and HuCCT1 cells using the PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea) and the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). The proteins were separated using SDS–PAGE with 10% Bis–Tris gels and transferred to membranes. The membranes were blocked with 5% skim milk and incubated overnight at 4°C with anti-KPNA2 rabbit polyclonal antibody (1:1000; Abcam), anti-MRE11 rabbit monoclonal antibody (1:2000; Abcam), anti-RAD50 mouse monoclonal antibody (1:2000; Abcam), anti-p95 NBS1 rabbit monoclonal antibody (1:2000; Abcam), GAPDH antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-β-actin mouse monoclonal antibody (1:1000; clone AC-74; Sigma, St. Louis, MO, USA). The membranes were then treated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Specific signals were detected using the ECL Prime Western Blotting Detection System (GE Healthcare, Tokyo, Japan) and quantified using an Image Quant LAS 4000 instrument (GE Healthcare).
3
Western Blot Analysis of Sperm Proteins
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Samples were lysed using RIPA lysis buffer, and protein concentrations were determined using a BCA Protein Assay Kit (23227; Thermo Fisher, Waltham, MA, United States). The proteins were extracted and separated by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (IPVH00010; Millipore, Billerica, MA, United States). Membranes were blocked for 1 h at room temperature with 5% skimmed milk in Tris-buffered saline solution (pH 7.4) containing 0.05% Tween-20 (TBST), and then incubated with anti-ACTRT1 (orb252388; Biorbyt, Cambridge, United Kingdom), anti-TSGA10 (12593-1-AP; Proteintech Group, Rosemont, IL, United States), anti-BRDT (AP7115a; Abgent, San Diego, CA, United States), or anti-acetylated tubulin (66200-1-Ig, Proteintech) primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (31460, Thermo Fisher) or goat anti-mouse IgG secondary antibody (31430, Thermo Fisher) for 1 h and washed three times with TBST at room temperature. The signals were developed using an enhanced chemiluminescence kit (K-12045-D50; Advansta, San Jose, CA, United States) and visualized and recorded using an ImageQuant LAS 4000 instrument (GE Healthcare Life Sciences, Marlborough, MA, United States).
4
Protein Extraction and Western Blot Analysis
5
Protein Expression Analysis Protocol
6
PAPP-A and STC2 Immunoprecipitation and Western Blot
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PAPP-A and STC2 immunoprecipitation was conducted exactly as described previously [29 (link), 40 (link), 41 (link)].No precipitation of target protein was seen with control IgG. Following elution of immune-precipitates proteins were separated by non-reducing 3–8% SDS-PAGE (1 h at 150 V) and transferred to PVDF membranes (Millipore). The blots were blocked for 5 min in 2% Tween 20 in ddH2O, then equilibrated in 50 mM Tris-HCl, 500 mM NaCl, 0.1% Tween 20, pH 9 (TST) and incubated overnight with primary antibodies (0.5–1.25 μg/ml) at room temperature. Antibodies were diluted in TST containing 2% skim milk (w/v). Blots were washed ×3 in TST, and incubated for 1 h at RT with secondary antibodies diluted in TST containing 2% skimmed milk (w/v). Blots were washed ×3 in TST and developed with enhanced chemiluminescence reagent (ECL Prime, GE Healthcare). Images were captured using an ImageQuant LAS 4000 instrument (GE Healthcare). Primary antibodies were rabbit polyclonal anti-PAPP-A at 1.25 μg/ml [40 (link)] and goat anti-STC1 at 0.5 μg/ml (R&D, AF2958). Secondary antibodies were Swine anti-rabbit IgG HRP (Dako, P0217) diluted 1:2000 and rabbit anti-goat IgG HRP (Dako, P0449) diluted 1:2000.
7
Quantification of Naïve and NEP-enhanced hUC-MSCs
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Naïve and NEP-enhanced hUC-MSCs were lysed with PRO-PREP™ Protein Extraction Solution (INtRON, Seongnam, Korea). Lysed cells were centrifuged at 13000 rpm at 4°C for 15 min to obtain a protein in the supernatant. The protein quantification was performed by using the Pierce™ BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA), incubated at 37°C for 30 min, and absorbance was measured at 562 nm with a set of albumin standards. Equal amounts of protein (50 μg) were loaded onto a 10% SDS gel, electrophoresed, and transferred to Immobilon-P PVDF membranes (Merck Millipore, Burlington, MA, USA). The protein was reacted with β-actin antibody (1 : 1000; Santa Cruz, TX, USA) or CD10 (NEP) antibody (1 : 1000; Santa Cruz, TX, USA) and then incubated with goat anti-mouse immunoglobulin (Ig) G antibody (HRP) (1 : 5000; Genetex, Irvine, CA, USA). The membrane was then washed and reacted using a Clarity Western ECL substrate (BIO-RAD, Hercules, CA, USA). Proteins were detected using an ImageQuant LAS 4000 instrument (GE Healthcare, Chicago, IL, USA). Experiments were independently replicated four times on different days. The detected protein was quantified by measuring the density using ImageJ.
8
Western Blotting with NuPAGE and Phospho-4EBP1 Detection
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Western blotting was performed using the NuPAGE SDS-PAGE gel system (Novex), according to the manufacturer’s protocol. The following antibodies were used at the dilutions indicated: anti-phospho-4EBP1 (Cell Signalling, 1:200) and anti-β-actin (Sigma Aldrich, 1:1,000). Secondary antibodies conjugated with horseradish peroxidase (HRP) were used at 1:50,000 dilution. The signal was developed with a SuperSignal West Femto Substrate kit (Thermo Scienctific) and visualized using an ImageQuant LAS 4000 instrument (GE Healthcare Life Sciences).
9
Western Blot Protein Analysis
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Cells (2 × 105) were seeded in 6-well plates and rested overnight before drug treatment for 48–72 hours. Whole-cell lysates were prepared using RIPA (10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with a protease inhibitor and a phosphatase inhibitor (both from Roche) and clarified by probe sonication and centrifugation (14,000g for 15 minutes at 4°C). Equal masses of protein (5 μg–40 μg) were separated by 4%–15% of SDS/PAGE and transferred onto nitrocellulose membranes (Bio-Rad) for protein blot analysis. Membranes were incubated with a primary antibody overnight and washed and incubated with a secondary antibody for 1 hour. Protein bands were visualized using either a fluorescence system (LI-COR) or Amersham ECL chemiluminescent reagent (GE Life Sciences); chemiluminescent signals were visualized with an ImageQuant LAS 4000 instrument (GE Healthcare).
10
Inducible Protein Expression in M. smegmatis
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10 ml 7H9 cultures supplemented with the suitable antibiotics were inoculated 1:25 with 7H9 precultures of M. smegmatis carrying pACE, pMEX or pMINT vectors and grown at 37 °C while shaking, until the cultures reached an OD600 of around 0.6. Cells were then induced with 100 ng/ml ATc (pMINT and pMEX) or 0.4% (w/v) acetamide (pACE) and grown overnight at 37 °C. 4 ml of cells were harvested in 2 ml tubes and resuspended in 350 μl 20 mM Tris/HCl pH 8, 200 mM NaCl. A spatula tip of acid-washed glass beads ≤106 µm (Sigma) was added and the cells were subsequently lysed with a FastPrep-24 Classic cell lysis machine (MP Biomedicals) for 3 cycles of 60 seconds with 6 m/s. The lysate was spun 15 minutes at 14’000 rpm at 4 °C with a F-45-30-15 rotor (Eppendorf) to pellet glass beads and cell debris. 40 μl of supernatant were mixed with 10 μl of 5 × SDS loading dye (120 mM TRIS pH 6.8, 50% glycerol, 100 mM DTT, 2% SDS (w/v), 0.1% bromophenol blue (w/v)), of which 10 µl were loaded on a 10% SDS-PAGE gel. The GFP-fusions were detected by in gel fluorescence with an ImageQuant LAS 4000 instrument (GE Healthcare). The exposure time was adjusted according to the intensity of the signal, and varied between 10 and 60 seconds.
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