SKOV3-SpyCatcher-BBζ cells were incubated with either 2000 nM myc-RFP-SpyTag or myc-RFP-SpyTag-DA for 1 h at 37 °C. Cells were lysed using RIPA lysis buffer with protease-inhibitor cocktail (Roche, cat. no. 5892970001) and centrifuged for 5 min. Lysate was then collected, and protein concentrations were quantified using a BCA assay (Thermo Scientific). Protein samples (80 μg) were mixed with loading buffer (Lammeli buffer; BioRad) containing 5% β-mercaptoethanol (BioRad) and incubated at 95 °C for 5 min. Samples were loaded in 4–15% Minigels protean TGX (BioRad) and run at 150 V for 1 h. A protein ladder (BioRad) was run along with the samples. Protein samples were transferred to a PVDF membrane (Millipore) at 100 V for 1 h. The membranes were washed with TBST (1% Tween; BioRad) and incubated with primary and secondary antibodies, including purified mouse anti-human CD3ζ (BD Pharmigen; 1:1000), anti-human/mouse/rat GAPDH, (R&D; 1:20 000), and peroxidase AffiniPure goat anti-mouse IgG (Jackson Immunology; 1:10 000). Membranes were washed three times in between the primary and secondary antibody incubation steps. Membranes were developed using the ECL prime Western blotting detection reagent (GE Healthcare no. RPN2236) and imaged using a GE ImageQuant LAS 4000 series imaging system.
Imagequant las 4000 series imaging system
Manufactured by GE Healthcare
The ImageQuant LAS 4000 series is a high-performance imaging system designed for life science research applications. It utilizes a charge-coupled device (CCD) camera technology to capture and analyze images of various samples, such as Western blots, gels, and membranes. The system provides sensitive and precise image acquisition, enabling researchers to visualize and quantify a wide range of biomolecular targets.
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Lab products found in correlation
Lammeli buffer, by Bio-Rad (2 mentions)
Minigels protean tgx, by Bio-Rad (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
β mercaptoethanol, by Bio-Rad (2 mentions)
Protein ladder, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Tbs t, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
4800 plus maldi tof tof analyzer, by AB Sciex (1 mentions)
Flp in t rex 293, by Thermo Fisher Scientific (1 mentions)
3 protocols using imagequant las 4000 series imaging system
1
Quantification of CD3ζ Protein Levels
2
Proteomic Analysis of GFP-Interacting Proteins
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Cell lysates of transiently transfected HEK293 cells from 90 % confluent 100 mm dish were harvested 40 h after transfection. Cell lysis and immunoprecipitation with GFP–Trap_A beads were performed as instructed by the manufacturer (ChromoTek). Input (corresponding to 2–3 × 105 cells), bound (corresponding to 1.2–1.5 × 106 cells in one lane) and unbound protein fractions were separated by 12 % SDS-PAGE and visualized with either Coomassie blue stain or Western blot. For MS analysis, bands of interest were excised and processed according to the method described in [50 (link)]. MS spectra were acquired on a 4800 Plus MALDI TOF/TOF analyzer (AB Sciex). Mascot server 2.2.07 and a current release of the SWISS-PROT human database were employed for peptide identification with the following settings: carbamidomethylation as fixed; methionine oxidation and N, Q deamination as variable modifications; one missed cleaving site allowed; precursor accuracy at 50 ppm; MS spectrum accuracy at 0.25 Da. Cell lines Flp-In™ T-REx™ 293 (Life Technologies) stably producing respective proteins were used for western blot analyses. Western blotting was performed as described previously [51 (link)], with the exceptions of using a PVDF membrane for protein transfer (Biorad) and the ImageQuant LAS4000 Series imaging system (GE Healthcare Life Sciences) for chemiluminescence signal acquiring.
3
Immunoblot Analysis of Anti-IL6Rα scFv-Fc
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