Lysate of PeV-1 infected HT-29 cells, purified VP0-GST fusion protein, HT-29 cell lysate and purified GST protein were heated in SDS sample buffer at 95 °C for 5 min, and run in 4–20% SDS PAGE (Bio-Rad 4–20% Mini-Protean® TGX™). Chameleon Duo (Licor 928-60000) was used as a marker. Separated proteins were transferred to membranes (Amersham™ Protran™ Nitrocellulose Blotting Membrane, Life Science #10600001) with TransBlot® Semi-Dry Transfer Cell (Bio-Rad). The blotted membranes were blocked overnight with TBS containing 5% BSA followed by blotting with 10 µg/ml of either scFv-55, -59 and -71 antibody in 1% BSA in TBS-T for 1 h. After washes with TBS-T, membranes were incubated with anti-FLAG antibody for 1 h. Membranes were then incubated with anti-rabbit secondary antibody (Licor IRDye® 800CW) for 1 h followed by washes and analysing with Odyssey (Licor). Full membranes were blotted and the area where all marker bands were visible was cropped together with the target band. Thus, any background binding is evident in the image. Experiments were repeated once.
Mini protean tgx
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The Mini-PROTEAN TGX is a precast polyacrylamide gel for protein electrophoresis. It is designed to provide consistent and reliable results in protein separation and analysis. The product features a Tris-Glycine-SDS buffer system and is available in various gel percentages to accommodate different protein size ranges.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (23 mentions)
Laemmli sample buffer, by Bio-Rad (15 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Chemidoc mp system, by Bio-Rad (9 mentions)
Chemidoc imaging system, by Bio-Rad (9 mentions)
G 21234, by Thermo Fisher Scientific (8 mentions)
Trans blot turbo transfer system, by Bio-Rad (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
Protease inhibitor cocktail, by Roche (7 mentions)
Odyssey fc imaging system, by LI COR (7 mentions)
Dc protein assay, by Bio-Rad (7 mentions)
Chemidoc mp imaging system, by Bio-Rad (7 mentions)
Cell lysis buffer, by Cell Signaling Technology (6 mentions)
Chemidoc xrs imaging system, by Bio-Rad (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (6 mentions)
Clarity western ecl substrate, by Bio-Rad (5 mentions)
Sc 32293, by Santa Cruz Biotechnology (5 mentions)
F21453, by Thermo Fisher Scientific (5 mentions)
242 protocols using mini protean tgx
1
Western Blot Analysis of PeV-1 VP0 Protein
2
Western Blot Analysis of BMI1 and H2AK119ub
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Cells without treatment or after incubation with 1.25 μM PTC-209 (72 h) were harvested by trypsin-EDTA treatment and counted. After centrifugation at 400 x g (3 minutes), the cell pellet was resupended in DPBS. Then cells were sonified (7-10 pulses, Sonopuls HD70, Bandelin, Berlin, Germany) and incubated for 5 minutes at 95°C in 2x SDS sample buffer. After that, 105 cells per sample were loaded on SDS gels (4-20% Mini-PROTEAN TGX, BioRad, Vienna, Austria). Western blot was performed with the Trans-Blot Turbo Mini Nitrocellulose Transfer Packs System (BioRad). Antibodies were used with the following concentrations: BMI1 (1:1000, Cell Signaling Technology, Danvers, Massachusetts, USA), H2AK119ub (1:300, Diagenode, Seraing, Belgium), BETA-ACTIN (1:1000, Cell Signaling Technology) and anti-rabbit IgG, HRP-linked (1:1000, Cell Signalling Technology). Membranes were developed with Signal Fire ECL Reagent (Cell Signaling Technology) and protein bands were detected and quantified using a ChemiDoc MP System (BioRad) and Image J, respectively.
3
1D SDS-PAGE Analysis of Plasma Proteins
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For each plasma pool, 10 μg of plasma (EP or whole plasma (WP)) were analysed in triplicates in order to expand protein identifications and to account for technical noise during data analysis [63 (link),69 (link)]. Samples were mixed in reducing buffer (13.1 mM Tris—pH 6.8, 2.63% v/v Glycerol, 0.42% v/v sodium dodecyl sulfate (SDS), 0.243% v/v bromophenol blue and 163.5 mM dithiothreitol (DTT)), heated up to 95°C for 5 min and centrifuged at 2,000 g for 30 s. Reduced lysates were loaded into a 1-D SDS polyacrylamide gel (4–15%, Mini-PROTEAN TGX, BIO-RAD) with a protein ladder reference (5μl, BenchMark, 10-220kDa, ThermoFisher Scientific). Gels were run using a Mini PROTEAN Tetra Cell System (Bio-Rad) at 200 V (400 mA) for around 50 min. Protein bands were fixed and stained with SimplyBlue Safestain (Thermo Fisher Scientific) following the manufacturer’s instructions and destained overnight in MilliQ water (Millipore, Merck) at RT. 1-D SDS-PAGE pictures were taken using an inGenius LHR Gel Imaging System (SynGene) and band densitometry data was obtained using GeneTools software version 4.3.8 (SynGene). Profile height (i.e. band intensity) to relative mobility (Rf) data was imported into R and plotted using ggplot2 package [70 ].
4
Western Blot Analysis of ApoE Fractions
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We used WB to detect ApoE positive fractions collected from the heparin binding columns. Fractions were diluted in 10 μL RIPA buffer (Cell Signaling Technology), 4 μL DTT (1 M, Sigma Aldrich) and 10 μl Laemmli buffer (Boston Bioproducts) to a final volume of 40 μl and denatured 5 min. at 60°C. Samples were separated electrophoretically for 1h at 70 V using 4–20% pre-cast gradient gels (Mini-PROTEAN® TGX™, Bio-Rad) and SDS-Tris-Glycine buffer. Proteins were transferred to nitrocellulose membranes (VWR; 27376–991) for 1 hour at 70 V. Membranes were blocked 1 hour with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE), and probed either 1 h room temperature (R.T.) or over-night at 4°C with anti-his tag antibody (rb, 1:5000, Novus biologicals), and 1 hour R.T. with IRDye 800CW donkey anti-rabbit (1:10000, LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using the Odyssey Infrared Imaging System and visualized on the Image Studio version 2.1 (LI-COR Biosciences). A composite of individual gels was used to generate Figure 2 . All western blotting reported in Figure 2 are representative of two independent experiments.
5
Western Blot Analysis of ApoE Fractions
6
Western Blot Analysis of Cell Signaling
7
Assessing CPT1a Expression in Jurkat Cells
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CPT shRNA knockdown Jurkat cells were lysed with 1× Laemmli Sample Buffer (Bio-Rad, #1610747) and the whole-cell lysates were fractionated by 4–20% SDS-PAGE (MINI-PROTEAN TGX, 4–20%, Bio-Rad) and transferred to a PVDF membrane. The membrane was incubated with antibodies against CPT1a (1 µg/ml, ab128568, Abcam) and GAPDH (1:2000, SC-25778, Santa Cruz) at 4 °C overnight, and subsequently incubated with 1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch) for 1 h at room temperature. The relative CPT expression level was determined by comparing to the control shRNA (NT#4) after first normalizing to GAPDH level.
For CPT1a protein expression, Jurkat cells were incubated with or without 200 µM C18:2 for 24 or 48 h, as indicated. The harvested cells were then lysed and the whole-cell lysates were subjected for western blot with antibodies against CPT1a (1 µg/ml, ab83862, Abcam) and GAPDH (1:2000, SC-343 25778, Santa Cruz) as described above.
For CPT1a protein expression, Jurkat cells were incubated with or without 200 µM C18:2 for 24 or 48 h, as indicated. The harvested cells were then lysed and the whole-cell lysates were subjected for western blot with antibodies against CPT1a (1 µg/ml, ab83862, Abcam) and GAPDH (1:2000, SC-343 25778, Santa Cruz) as described above.
8
SDS-PAGE Protein Profiling of Membrane Streams
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Protein profile of feed, retentate, and permeate streams was qualitatively assessed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) with precast gels (Mini-PROTEAN TGX, Bio-Rad Laboratories, Irvine, CA, USA) under reducing conditions using an AcquaTank mini gel unit (Acquascience, Bellbrook Industrial Estate, Uckfield, UK) [2 (link)]. The final protein concentration of 1 mg/mL for feed and retentate samples was used for loading, and permeate was used as is. The sample solution of 8 μL was loaded for feed and retentate samples, while 20 μL was used for permeate samples, and all gels were Coomassie-stained. SDS-PAGE gels were scanned using a desktop scanner (HP Scanjet G4010, HP, Leixlip, Ireland) and densitometry was performed using GelAnalyzer 19.1 (www.gelanalyzer.com ) by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc.
9
SDS-PAGE Analysis of Host Cell Protein Release
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The release of host cell proteins by electroporation was measured in triplicate by SDS-PAGE. PEF extracts and reference sample were diluted 10× and 3×, respectively, in dH2O. Samples were further diluted in 2 × Laemmli buffer and incubated at 95°C for 10 min. Samples (10 μl) were then loaded onto precast SDS gels (4–15%, Mini-PROTEAN TGX; Bio-Rad, Hercules, CA), which were run at 180 V for 30 min. After staining with Coomassie Blue, the gels were imaged with a ChemiDoc system (Bio-Rad) and densitometric analysis was done with the GelAnalyzer software (version 19.1)1 . The impurity load in percent was consequently calculated according to Eq. 4 using the area from the integrated curves of the densitograms:
10
Native PAGE Analysis of α2-Macroglobulin Purification
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Purified human α2-macroglobulin protein (725 kDa) was obtained from Enzo Life Science (New York, USA), anti-human monoclonal mouse α2-macroglobulin (immunoglobulin (Ig)G1 clone) from R&D Systems (Minneapolis, MN, USA), and goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) conjugate from Bio-Rad Laboratories (Hercules, CA, USA). Native PAGE™ Sample Buffer, Native PAGE Running Buffer, Dark Blue Cathode Buffer, Native Mark™ Unstained Protein Standard, and Light Blue Cathode Buffer were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DTT, Blocking One and Peroxidase Stain 3,3′-Diaminobenzidine kit (Brown Stain) were purchased from Nacalai Tesque (Kyoto, Japan). Immune-Blot® polyvinylidene difluoride membrane, Sequi-Blot™ membrane, Precision Plus Protein™ Dual Color Standards and Mini-PROTEAN® TGX™ were obtained from Bio-Rad Laboratories. Perfect NT Gel, Perfect NT Gel System and SDS-PAGE Running Buffer were obtained from DRC (Tokyo, Japan), and ECL Prime Western Blotting Detection Reagent from GE Healthcare (Buckinghamshire, UK).
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