Cultured cells were collected in lysis buffer (5 mmol·L-1 Tris, 5 mmol·L-1 EDTA/EGTA, and proteinase inhibitors) and then ruptured by pipetting through a 20 gauge needle. Cell lysates were then centrifuged at 45 000 × g for 45 min. The pellet was resuspended in lysis buffer and the membrane protein was dissolved by addition of sodium dodecyl sulfate (SDS) to a 1% final concentration. Protein concentrations of SDS-dissolved lysates were determined by Micro BCA Protein Kit (Thermo Scientific, Rockford, IL, USA) and the lysates were used for Western blotting analysis. Each protein sample was boiled in SDS loading buffer, subjected to electrophoresis on a 10% SDS-polyacrylamide gel, and electroblotted onto a nitrocellulose membrane. Membranes were incubated with a 1:300 dilution of affinity-purified anti-Cx43 antibody,73 (link) or a 1:5 000 dilution of monoclonal anti-β-actin antibody (Sigma). Primary antibodies were detected with goat anti-rabbit IgG conjugated IRDye® 800CW and goat anti-mouse IgG conjugated IRDye® 680RD (1:15 000 dilution) using a Licor Odyssey Infrared Imager (Lincoln, NE, USA), as previously described.74 (link) The band intensity was quantified by densitometry using Image J software (NIH, Bethesda, MD, USA).
Micro bca protein kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Micro BCA Protein Assay Kit is a colorimetric detection method used to quantify total protein concentration in small sample volumes. The kit utilizes bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. The method combines the well-known reduction of Cu2+ to Cu+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid.
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Lab products found in correlation
Rc dc protein assay kit, by Bio-Rad (2 mentions)
Trans blot turbo transfer pack, by Bio-Rad (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Pegde, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Alp kit, by Merck Group (1 mentions)
Wst 1 kit, by Cayman Chemical (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
H2so4, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
10 protocols using micro bca protein kit
1
Membrane Protein Extraction and Western Blot Analysis
2
Biomaterial Synthesis Protocol
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Pure grade acetone, hexane,
PEGDE, NaOH, H3PO4, H2SO4, C2H2O4 (oxalic acid), β-glycerophosphate,
Trypsin-EDTA, Trypan blue, Alizarin Red S, Penicillin–streptomycin,
Fetal calf serum, DMEM, and ALP kit were purchased from Sigma-Aldrich.
High purity Al foils (99.999%, Puratronic, 1 mm thickness) were obtained
from Alfa Aesar, and Si ⟨111⟩ wafer was obtained from
Micro Chemicals GmbH. Collagen and Gelatin were purchased from NeoCell
Super Collagen and Halavet Food, Industry and Trade Inc., respectively.
Micro BCA protein kit and DAPI were obtained from Thermo Scientific.
ProteinEX was received from Gene All; PBS (10×) was purchased
from Amresco;l -glutamine was obtained from Gibco; WST-1
kit was purchased from Cayman Chemical.
PEGDE, NaOH, H3PO4, H2SO4, C2H2O4 (oxalic acid), β-glycerophosphate,
Trypsin-EDTA, Trypan blue, Alizarin Red S, Penicillin–streptomycin,
Fetal calf serum, DMEM, and ALP kit were purchased from Sigma-Aldrich.
High purity Al foils (99.999%, Puratronic, 1 mm thickness) were obtained
from Alfa Aesar, and Si ⟨111⟩ wafer was obtained from
Micro Chemicals GmbH. Collagen and Gelatin were purchased from NeoCell
Super Collagen and Halavet Food, Industry and Trade Inc., respectively.
Micro BCA protein kit and DAPI were obtained from Thermo Scientific.
ProteinEX was received from Gene All; PBS (10×) was purchased
from Amresco;
kit was purchased from Cayman Chemical.
3
Isolation and Purification of Gonococcal Outer Membranes
4
Western Blot Analysis of Protein Lysates
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Protein lysates from cells were obtained by lysis in radioimmunoprecipitation assay buffer (Merck Millipore, R0278) with protease and phosphatase inhibitors (Roche, 04906837001). A Micro BCA Protein kit (ThermoFisher, 23235) was used to measure protein levels, and equal amounts of proteins were resolved by electrophoresis on 12% Tris-HCl gels (Mini-PROTEAN TGX gels; Bio-Rad, 4561043) and transferred onto polyvinylidene difluoride membranes (Trans-Blot Turbo Transfer Pack; Bio-Rad, 1704156). Membranes were developed using the indicated primary antibody at a 1:1,000 dilution and secondary antibodies at a 1:3,000 dilution in blocking solution. This was followed by incubation with chemiluminescent HRP detection reagent (Merck Millipore, WBKLS0500) for 1 min before image acquisition by the ChemiDoc Imaging System (Bio-rad).
5
Determining Encapsulation Efficiency of PLGA+Chitosan+IL-10 Nanoparticles
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The percent EE was determined as reported previously.37 ,39–42 (link) Briefly, 20 mg of PLGA+chitosan+IL-10 nanoparticles were mixed with NaOH and sodium dodecyl-sulfate (SDS) overnight on a rocker at room temperature (RT). Nanoparticles were centrifuged, and supernatants were obtained to measure the released IL-10 using the microBCA protein kit (ThermoFisher, Waltham, MA). EE calculation is shown below with total IL-10 (A) and non-encapsulated IL-10 (B).
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$${{\rm EE\ }}\left({{\rm \% }} \right){{\rm \ =}}\left[{{{{{\rm A - B}}} \over {{\rm A}}}} \right]{{\rm \ x\ 100}}$$
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$${{\rm EE\ }}\left({{\rm \% }} \right){{\rm \ =}}\left[{{{{{\rm A - B}}} \over {{\rm A}}}} \right]{{\rm \ x\ 100}}$$
\end{document}
6
Isolation and Purification of Gonococcal Outer Membranes
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After 18–22 h culture on supplemented GC agar, N. gonorrhoeae was harvested from plates into ice-cold lithium acetate buffer (pH 5.8) and passed through a 25-gauge needle 10–12 times to sheer the outer membranes from the bacteria. The suspensions were spun in microfuge tubes at 13,000 RPM for 1 min. The supernatants were collected and ultracentrifuged at 107,000×g for 2 h. The pellet was washed with 50 mM Tris-HCl (pH 8.0) and resuspended in PBS. Protein was assayed with the Micro BCA protein kit (Thermo Scientific, Rockford, IL) or RC DC Protein Assay kit (Bio-Rad, Hercules, CA).
7
Deglycosylation Analysis of Bone Proteoglycans
8
Enzyme Adsorption Kinetics on PET
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The adsorption of the enzymes to PET was determined using a fixed PET concentration of 50 g·L−1 and enzyme concentrations ranging from 0 to 1.5 µM. PET reactions were incubated in low binding 96‐well plates for 1 h and 1000 rpm at either 50 or 60°C. The separation of the solid and liquid phase was done by centrifugation in a preheated centrifuge (set to the incubation temperature). The protein content of the supernatant after incubation with PET was determined using a micro BCA protein kit from Thermo Fischer Scientific (Product No.: 23225), where 100 µl supernatant was mixed with 100 µl freshly prepared BCA working solution. The subsequent color reaction took place in a microtiter plate incubated at 37°C for 2 h and 300 rpm. After incubation with the BCA reagent, 150 µl was transferred to a new microtiter plate and the absorbance was read at 562 nm in a plate reader. Standard curves of the individual enzymes (ranging from 0 to 1.5 µM in concentration) were used to quantify the amount of free enzyme in the reactions. From the amount of free enzymes (Efree) and the total enzyme concentration (E0), the amount of bound enzyme could be calculated as Ebound = E0 – Efree. Data were fitted to a Langmuir isotherm and adsorption parameters extracted using ORIGIN PRO 2019. All experiments were done in triplicates.
9
Protein Expression Analysis by Western Blot
10
Mitochondrial Protein Solubilization and Separation
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Protein concentrations were determined using the microBCA protein kit (Thermo Scientific). Mitochondria were solubilized with 6 g digitonin (Sigma) per gram of protein in 50 mM NaCl, 5 mM 6-aminohexanoic acid, 2 mM EDTA, and 50 mM imidazole/HCl (pH 7.0). Blue native electrophoresis was performed as described in Wittig et al. (2006) .
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