The quality and purity of immunoglobulins Y from SC were identified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was conducted under reducing and denaturing conditions with 7% stacking gel and 12% separation gel on an electrophoresis device (Bio-Rad Laboratories, Hercules, CA, USA). The protein bands were stained with Coomassie brilliant blue (CBB) (Bio-safe Coomassie, Bio-Rad, Hercules, CA, USA). The gel was analyzed measuring density after background subtraction using Image J.JS ImJoy gel analysis tool (http://rsbweb.nih.gov/ij/index.html ). IgY antibody activity was evaluated with ELISA.
Electrophoresis device
Manufactured by Bio-Rad
Sourced in United States
The Electrophoresis device is a laboratory instrument used for the separation and analysis of macromolecules, such as proteins, nucleic acids, or other charged particles, based on their size and/or charge. The device creates an electric field that allows the migration of these molecules through a gel or buffer solution, enabling their separation and identification.
Automatically generated - may contain errors
Lab products found in correlation
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Anti p2x7, by Alomone (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Caspase 1, by ABclonal (1 mentions)
Biacore t200, by GE Healthcare (1 mentions)
Epoch 2 microplate reader, by Agilent Technologies (1 mentions)
Cf3cooh, by Merck Group (1 mentions)
Xevo g2 xs qtof, by Waters Corporation (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
Griess reagent, by Beyotime (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Myd88, by ABclonal (1 mentions)
Nlrp3, by ABclonal (1 mentions)
Lambda ladder pfg marker, by New England Biolabs (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
A32731, by Thermo Fisher Scientific (1 mentions)
7 protocols using electrophoresis device
1
Characterizing IgY antibody purity
2
OM-PLA1 Cloning and Expression in P. pastoris
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According to the gene sequences of OM-PLA1 in the NCBI database (HG326223.1), primer pair Us-OM-PLA1 and Ds-OM-PLA1 was synthesized to amplify the S. marcescens genome for OM-PLA1 cloning using PCR amplification instrument (TOMOS, United States) (Table 1 ). A phylogenetic tree was drawn by using Lagergene MegAlign software. The OM-PLA1 gene was inserted into plasmid pEASY-E1. The recombinant plasmid was named pEASY-OM-PLA1 after sequencing confirmation. Then, pEASY-OM-PLA1 was used to amplify OM-PLA1 with upstream primer Us-SnabI-OM-PLA1 and downstream primer Ds-AvrII-OM-PLA1. OM-PLA1 carrying cohesive ends was inserted into the pPIC9K plasmid digested by SnabI and AvrII (Gohel and Singh, 2012 (link); Gamerith et al., 2017 (link)). The recombinant plasmid was named pPIC9K-OM-PLA1 by sequencing confirmation. pPIC9K-OM-PLA1 was transformed into P. pastoris GS115 by electroporation (Guo et al., 2015 (link)). The electrophoresis device and gel imaging system for DNA test were from Bio-Rad Company (United States).
3
Microglial Protein Extraction and Western Blot
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After reagent treatment for 24 h, primary cultured microglia seeded on 6-well plates were lysed in RIPA buffer (Beyotime Biotechnology, China) for protein extraction. The composition of RIPA buffer was as follows: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA and leupeptin. The lysate was centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected and preserved at -20°C for later use. The protein content of the samples was determined via the Lowry method. Samples containing equal amounts of protein (20 μg) were diluted with sample buffer, heated to 95°C for 10 min, and separated on SDS-polyacrylamide gels (10%) using an electrophoresis device (Bio-Rad, USA). Proteins were transferred onto PVDF membranes. A multifunctional gel imaging system was used to detect chemiluminescent signals. Band density was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The primary antibodies were as follows: rabbit polyclonal anti-P2X7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution). The secondary antibody was goat anti-rabbit/mouse IgG (Beijing Zhongshan Biotech Co.). Band densities were normalized with β-actin internal controls.
4
Evaluating PEI-AuNPs Conjugation with miR-3074-3p
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About 20 μM antagomiR-NC or antagomiR-3074-3p was mixed with PEI-AuNPs solution
at different volume ratios. The ability of PEI-AuNPs to conjugate with
miR-3074-3p was evaluated by conducting 1% agarose gel electrophoresis at room
temperature for 15 min using an electrophoresis device (Bio-rad, America).
at different volume ratios. The ability of PEI-AuNPs to conjugate with
miR-3074-3p was evaluated by conducting 1% agarose gel electrophoresis at room
temperature for 15 min using an electrophoresis device (Bio-rad, America).
5
Molecular Mechanisms of Neuroinflammation
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DMEM was purchased from Hyclone (Beijing, China). Fetal bovine serum (FBS) was purchased from BI (Israel). Griess reagent was purchased from Beyotime (Shanghai, China). LPS, RIPA buffer, and CF3COOH (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit antibodies against mouse iNOS, COX-2, Tau, p-Tau, IBA-1 TREM2, DAP12, TLR4, MyD88, Caspase-1, and NLRP3 were obtained from Abclonal (Wuhan, China). HPLC-grade methanol and acetonitrile were supplied from Merck KGaA (Darmstadt, Germany). PBS, CM5 sensing chips were obtained from from GE Healthcare (Sweden). HP-20 macroporous resin was purchased from Mitsubishi Chemical Company (Japan).
An ultrasonic cleaner, high-speed freezing centrifuge, and electric heater were obtained from Branson (EYELA, Tokyo, Japan), and Biacore T200 (GE Healthcare, Sweden). The Xevo G2-XS Qtof was acquired from Waters Inc (Milford, USA.). Deionized water was collected from a Milli-Qwater purification system (Merck KGaA, Darmstadt, Germany). The rotavapor tandem cold trap was from EYELA (EYELA, Tokyo, Japan). The Epoch 2 microplate reader was got by BioTek (USA). The Electrophoresis Device and scanning imager were acquired from BIO-RAD.
An ultrasonic cleaner, high-speed freezing centrifuge, and electric heater were obtained from Branson (EYELA, Tokyo, Japan), and Biacore T200 (GE Healthcare, Sweden). The Xevo G2-XS Qtof was acquired from Waters Inc (Milford, USA.). Deionized water was collected from a Milli-Qwater purification system (Merck KGaA, Darmstadt, Germany). The rotavapor tandem cold trap was from EYELA (EYELA, Tokyo, Japan). The Epoch 2 microplate reader was got by BioTek (USA). The Electrophoresis Device and scanning imager were acquired from BIO-RAD.
6
PFGE Genotyping of S. aureus Isolates
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All S. aureus isolates including NCTC 8325 reference strain were subjected to genotyping using PFGE according to previously published procedures (Delgado et al. 2011) . Briefly, bacterial DNA was subjected to digestion using SmaI endonuclease enzyme (New England Biolabs, USA) at 37°C for 18 hours. The electrophoresis was then carried out using electrophoresis device (Bio-Rad, USA) for 23 hours at 14°C at 6V/cm with 5 to 50 second pulses with an angel of 120u and a linear ramping factor. A standard pattern was included in the gels to allow comparison of the digitally normalized PFGE profiles (Lambda Ladder PFG Marker, New England Biolabs, USA). Obtained gels were stained and images (containing 84 bacterial isolates patterns) were obtained and analyzed using computer software (PyElph 1.4, Informer Technologies, Inc., USA). The dice coefficient represented by unweighted pair group using the arithmetic averages (UPGMA) clustering method with 1% band position tolerance and 0.5% optimization settings was used to determine clusters (Aklilu et al. 2012 (link)). An 80% cut-off similarity and criterion of a difference of #6 bands were both used to define a cluster (Aklilu et al. 2012) (link). Isolate information and a dendrogram were created to estimate the significant genetic variation/similarities between the bacterial isolates using 22 representative isolates.
7
Protein Expression Analysis in Cell Lines
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The expression of TLR2, TLR4, MyD88, G-CSF, IL-12 and IL-6 in each cell line was detected by Westernblot method. 40 μg of samples were added with 10% SDS-PAGE buffer and electrophoresed using Bio-Rad electrophoresis device. Then they were transferred to the PVDF membrane for 30-60 mins, blocked, and incubated with the following primary antibody overnight: anti-rabbit TLR2 (1:1000, PA5-17492, ThermoFisher, Shanghai, China), TLR4 (1:200, 48-2300, ThermoFisher), MyD88 (2 µg/mL, PA5-19919, ThermoFisher), G-CSF (1:1000, PA5-86821, ThermoFisher), IL-12 (0.8µg/mL, PA5-18741, ThermoFisher), IL-6 (1:1000, P620, ThermoFisher) and β-actin (1:8000, PA1-16889, ThermoFisher). After rewarming, the membranes were incubated with secondary antibody Ig G (0.3µg/mL, A32731, ThermoFisher) at room temperature for 1 h. Then they were washed and colored with ECL luminescent substrate for 3-5 mins.
Protein expression levels were scanned, quanti ed and nally normalized relative to β-actin using the Image J (NIH) software.
Protein expression levels were scanned, quanti ed and nally normalized relative to β-actin using the Image J (NIH) software.
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