Purified proteins were mixed with loading buffer and loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were electrophoresed for 110 min at 80 V and 500 mA and visualized using Coomassie Brilliant Blue G250 staining (Sigma-Aldrich) or transferred to a nitrocellulose blotting membrane (Sigma-Aldrich) at a constant current of 250 mA for 90 min. Next, the membranes were blocked in 0.5% bovine serum albumin (BSA) (Sigma-Aldrich) and incubated with human anti-S1 antibody (Abcam, Cambridge, UK) for 3 h at room temperature. After being washed, the membranes were incubated with horseradish peroxidase (HRP)-linked rabbit anti-human IgG antibody (Abcam) for 1 h at room temperature. Membranes were washed three times with PBST [1× phosphate-buffered saline (PBS), 0.1% Tween® 20 Detergent], and the protein bands were visualized by enhancing the TMB (3,3′, 5,5′-tetramethylbenzidine) substrate solution (Sigma-Aldrich).
Coomassie brilliant blue g 250 staining
Manufactured by Merck Group
Sourced in Germany
Coomassie Brilliant Blue G-250 is a laboratory staining dye used to detect and quantify proteins in various analytical techniques. It binds to basic and aromatic amino acid residues, causing a color change that can be measured spectrophotometrically. This dye is commonly used in protein assays, such as the Bradford assay, to determine protein concentration in a sample.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine tmb substrate solution, by Merck Group (1 mentions)
Nitrocellulose blotting membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Human anti s1 antibody, by Abcam (1 mentions)
Phenol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Amplex red reagent, by Thermo Fisher Scientific (1 mentions)
2 6 dichloroindophenol, by Merck Group (1 mentions)
3 5 dinitrosalicylic acid, by Merck Group (1 mentions)
S3139, by Merck Group (1 mentions)
G7141, by Merck Group (1 mentions)
S9763, by Merck Group (1 mentions)
S0505, by Merck Group (1 mentions)
F7250, by Merck Group (1 mentions)
Sephadex g 50 medium, by Merck Group (1 mentions)
3 protocols using coomassie brilliant blue g 250 staining
1
SDS-PAGE and Western Blot Analysis
2
Heterologous expression of MtCDHA gene
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The MtCDHA gene (MYCTH_111388) from Myceliophthora thermophila M77 was PCR amplified from genomic DNA without the original signal peptide. The PCR product was amplified using the oligonucleotide primers: forward (5'-gggttggcaCAGAACAACGCGCCGGTAACCTTCACCGAC-3') and reverse
(5'gtcccgtgccggttaTCACAAGCACTGCGAGTACCACTCGTTCTGCATCTGGCACGT-3')
and was cloned into the pEXPYR vector using the Ligation-Independent Cloning protocol (LIC). 21 The expression plasmid was transformed in A. nidulans A773 (pyrG89; wA3; pyroA4) as described earlier. 22 Approximately 10 7 spores/mL were inoculated in liquid minimal medium at pH 6. Coomassie Brilliant Blue G-250 staining (Sigma, Deisenhofen, Germany).
(5'gtcccgtgccggttaTCACAAGCACTGCGAGTACCACTCGTTCTGCATCTGGCACGT-3')
and was cloned into the pEXPYR vector using the Ligation-Independent Cloning protocol (LIC). 21 The expression plasmid was transformed in A. nidulans A773 (pyrG89; wA3; pyroA4) as described earlier. 22 Approximately 10 7 spores/mL were inoculated in liquid minimal medium at pH 6. Coomassie Brilliant Blue G-250 staining (Sigma, Deisenhofen, Germany).
3
Enzymatic Colorimetric Assays for Biomolecules
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Hydrogen peroxide 30 wt.% (VWR Chemicals), horseradish peroxidase ~150 U/mg (77332, Sigma-Aldrich), absolute ethanol (VWR Chemicals, ≥99.8), microcrystalline cellulose
(BASF Basionics, ≥95%), DMSO (Alfa Aesar, ≥99%), glucose oxidase from Aspergillus niger (G7141, Sigma-Aldrich) (AnGOx), bovine serum albumin (05470, Sigma-Aldrich, ≥96%), Sodium iodide (383112, Sigma-Aldrich, ≥99.5%), sodium phosphate dibasic (S9763, Sigma-Aldrich, ≥99%), sodium phosphate monobasic (S3139, Sigma-Aldrich), sodium carbonate (S7795, Sigma-Aldrich, ≥98%), sodium bicarbonate (S5761, Sigma-Aldrich, ≥99.5%), sodium sulfite (S0505, Sigma-Aldrich, ≥98%), Phenol (328111, Sigma-Aldrich, ≥99%), Amplex ® Red reagent (Invitrogen), 2,6-DichloroindoPhenol (D1878, Sigma-Aldrich), Sephadex G-50 Medium (Sigma-Aldrich), 3,5-Dinitrosalicylic acid (D0550, Sigma-Aldrich, ≥98%), Coomassie Brilliant Blue G-250 staining (Sigma, Deisenhofen, Germany), fluorescein isothiocyanate (F7250, Sigma-Aldrich, ≥90%).
(BASF Basionics, ≥95%), DMSO (Alfa Aesar, ≥99%), glucose oxidase from Aspergillus niger (G7141, Sigma-Aldrich) (AnGOx), bovine serum albumin (05470, Sigma-Aldrich, ≥96%), Sodium iodide (383112, Sigma-Aldrich, ≥99.5%), sodium phosphate dibasic (S9763, Sigma-Aldrich, ≥99%), sodium phosphate monobasic (S3139, Sigma-Aldrich), sodium carbonate (S7795, Sigma-Aldrich, ≥98%), sodium bicarbonate (S5761, Sigma-Aldrich, ≥99.5%), sodium sulfite (S0505, Sigma-Aldrich, ≥98%), Phenol (328111, Sigma-Aldrich, ≥99%), Amplex ® Red reagent (Invitrogen), 2,6-DichloroindoPhenol (D1878, Sigma-Aldrich), Sephadex G-50 Medium (Sigma-Aldrich), 3,5-Dinitrosalicylic acid (D0550, Sigma-Aldrich, ≥98%), Coomassie Brilliant Blue G-250 staining (Sigma, Deisenhofen, Germany), fluorescein isothiocyanate (F7250, Sigma-Aldrich, ≥90%).
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