The method of protein expression and purification was previously described (Zhang et al., 2020 (link)). A BL21(pETmclX) strain was grown in LB liquid medium until reaching an OD600 of 0.7. Then, 1 mM isopropyl-beta-D-thiogalactoside (Solarbio, Beijing, China) was added to induce MclX-His expression, and bacteria were harvested after overnight growth at 18°C and sonicated (CP750, Cole-Parmer). The supernatant was loaded onto a 2-ml suspension of Ni Sepharose 6 Fast Flow (GE Healthcare, Sweden) with wash buffer (25 mM Tris-HCl, 50 mM imidazole, 0.5 M NaCl, pH 8.0) and elution buffer (25 mM Tris-HCl, 250 mM imidazole, 0.5 M NaCl, pH 8.0). The purified MclX-His protein was analyzed by 10% SDS–PAGE (Mini protein III, Bio-rad).
Mini protein 3
Manufactured by Bio-Rad
Sourced in Germany
The Mini-Protein III is a compact and versatile electrophoresis system designed for small-format polyacrylamide gel electrophoresis. It is suitable for resolving proteins under denaturing or native conditions.
Automatically generated - may contain errors
Lab products found in correlation
Ni sepharose 6 fast flow, by GE Healthcare (1 mentions)
Isopropyl beta d thiogalactoside, by Solarbio (1 mentions)
Cp750, by Cole-Parmer (1 mentions)
Gs 710 calibrated imaging densitometer, by Bio-Rad (1 mentions)
Hyperfilm ecl, by Cytiva (1 mentions)
Ecl plus kit, by Cytiva (1 mentions)
Quantity one quantitation software, by Bio-Rad (1 mentions)
Hybond p, by Cytiva (1 mentions)
4 protocols using mini protein 3
1
MclX-His Protein Expression and Purification
2
Quantification of Hepatic CYP2E1 Protein
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Hepatic CYP2E1 protein expression was determined as described previously [23 (link)]. Briefly, hepatic microsomal proteins (5 μg) were denatured with sample buffer, loaded on a 10 % (w/v) SDS-polyacrylamide gel, and then electrophoresed (Mini-Protein III, Bio-Rad, CA). The gel contents were electrophoretically transferred to a PVDF-membrane (Hybond™-P, Amersham). The membrane was blocked in non-fat skimmed milk and incubated at 4 °C overnight with primary antibody at a 1:20,000 dilution (rabbit anti-rat CYP2E1, Chemicon International, Inc., Temecula, CA). After several washes in 0.1 % Tris Buffered Saline with Tween-20, the membranes were incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Chemicon) at room temperature for 1 h. The ECL Plus kit (Amersham) was used for the chemiluminescence reaction and exposure to Hyperfilm ECL™ (Amersham). GS-710 Calibrated-Imaging densitometer and Quantity One Quantitation software (Bio-Rad, Hercules, CA) were used for CYP2E1 protein analysis.
3
Visualizing Larval Sarcophagidae Salivary Gland Proteins
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Larval SGLs of L. sericata were extracted, and the proteins and/or glycoproteins were visualized by SDS-PAGE on 1-mm-thick 12.5% Tris-glycine gel with 110-V fixed voltage using “Mini-Protein III” (Bio-Rad, Munich, Germany) under reducing conditions.
The SGLs from three to five pooled glands were loaded into each well. Following electrophoresis, the gels were stained with silver nitrate according to the methods described by Heukeshoven and Dernick [35 (link)]. A pre-stained protein ladder (PageRuler, Fermentas) was used to estimate the molecular weights of the protein bands.
The SGLs from three to five pooled glands were loaded into each well. Following electrophoresis, the gels were stained with silver nitrate according to the methods described by Heukeshoven and Dernick [35 (link)]. A pre-stained protein ladder (PageRuler, Fermentas) was used to estimate the molecular weights of the protein bands.
4
Protein Profile Analysis of Larval ES
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The protein profile of the crude and fractionated larval ES (>10 and <10 kDa) of L. sericata were analyzed using 1-mm-thick 12.5% Sodium dodecyl sulfate–polyacrylamide gel at a 110-V constant voltage using “Mini-Protein III” system (Bio-Rad) under reduction conditions. The gels were then stained with a solution containing Coomassie brilliant blue G250 and methanol. The molecular weights of the protein bands were estimated in comparison with a prestained protein ladder (PAGEmark, 786–418). About 20–25 protein bands were observed for crude ES, which corresponded to bands in both the >10 kDa and <10 kDa fractions. The protein profiles of larval ES and its fractions were presented in our previous study [30 (link)]. The profile obtained from ES separation showed that the separation was done with high accuracy.
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