DTNB (Ellman's Reagent) (5,5-dithio-bis-(2-nitrobenzoic acid) (Thermo Fisher Scientific) was dissolved in 0.1 M sodium phosphate buffer, pH 8.0 to 1 mM. Recombinant A1M (130 μM) was incubated with different ratios of HS6 for 30 min at RT. Eight standard samples were prepared with cysteine hydrochloride monohydrate (Sigma-Aldrich) in a range between 0 μM and 1500 μM 100 μl of each sample was added in duplicates to a 96-well plate followed by addition of 100 μl DTNB (1 mM). The plate was incubated for 15 min at RT covered in aluminum foil and absorbance was measured at 405 nm for 0.1 s (VICTOR 1420 multilabel reader, PerkinElmer). A standard curve was calculated (sigmoidal, 4 PL) and the amount of free thiol in each sample was determined. The amount of free thiol in the A1M only sample was set to 100%. The true value for the amount free thiol in the A1M only sample was approximately 42%, which is within the range found in previous studies [28 (link)].
Cysteine hydrochloride monohydrate
Manufactured by Merck Group
Sourced in United States
Cysteine hydrochloride monohydrate is a chemical compound that is used as a laboratory reagent. It is a crystalline solid that is soluble in water and has a characteristic odor. The compound is typically used in various analytical and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ellman s reagent, by Merck Group (2 mentions)
Victor 1420 multilabel reader, by PerkinElmer (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Ellman s reagent, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Cellulose membrane, by Merck Group (1 mentions)
Low density lipoprotein, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
4 protocols using cysteine hydrochloride monohydrate
1
Quantification of Free Thiols in A1M
2
Quantifying Sulfhydryl Groups Using Ellman's Assay
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Samples were mixed 1:10 into an 80 µg/ml solution of Ellman’s Reagent (Thermo Scientific Pierce, Waltham, MA, USA) diluted in reaction buffer (0.1 M sodium phosphate with 1 mM EDTA, pH 8.0) and allowed to incubate at room temperature for 15 min. A standard curve was generated using cysteine hydrochloride monohydrate (Sigma Aldrich, St. Louis, MO, USA) at concentrations ranging from 0.25 to 1.5 mM in the reaction buffer mixed with the Ellman’s Reagent at the same proportion and incubated the same as the samples. Samples and standards were read at an absorbance of 412 nm in a BioTek Synergy HT plate reader and a linear regression was generated and used to analyze the samples yielding a calculation of mM sulfhydryl groups relative to a cysteine standard.
3
Quantification of Free Cellular Thiols
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The quantification of free cellular thiol was performed according to Ellman [63 (link)] and Riddles et al. [64 (link)], with modifications. For each 25 μL of the supernatant or standard, 5 μL of 0.4% (w/v) 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB or Ellman’s reagent; Sigma, USA) in buffer sodium phosphate 0.1 M pH 8.0 containing 1mM EDTA and 250 μL of the buffer sodium phosphate were added in microplate. The microplate was incubated at room temperature for 15 min and the absorbance at 412 nm measured by using a spectrophotometer (Thermo Fisher Scientific, Finland). The free cellular thiol was quantified by using cysteine hydrochloride monohydrate (Sigma, USA) as standard at concentrations from 0.0 to 1.5 mM. The obtained equation was as follows: absorbance = (0.9421 x concentration) + 0.0432, with R2 = 0.9936. Next, the quantification of free cellular thiol was normalized by OD600nm.
4
Fluorescent Antibody and Protein Labeling
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Fluorescein isothiocyanate (FITC) conjugated anti-CWP1 monoclonal antibody (mAb) was purchased from Waterborne Inc. (New Orleans, LA, USA). 2F5 mAb was used for the μ2 subunit of AP-223 (link). Anti-VSP1267 5C1 mAb was a gift of Dr. Nash70 (link). Anti-HA mAb, Apo-lactoferrin containing 4.1% iron, from bovine milk (bLF), poly-L-Lysine, formaldehyde, cysteine hydrochloride monohydrate, bovine serum albumin and low-density lipoprotein (LDL) from human plasma were purchased from Sigma-Aldrich (St. Louis, MO). FluorSave™ reagent was from Calbiochem (San Diego, CA). Human chylomicrons were acquired from Athens Research & Technology (Athens, GA, USA). LysoTracker™ Red DND-99 used to label PVs was acquired from Molecular Probes-Invitrogen (Carlsbad, CA). FITC-(N-Terminal) bLFcin (FKCRRWQWRMKKLGAPSITCVRRAF) was synthesized and labelled by GenScript® (Piscataway, NJ, USA). For FITC-Labeling bLF, 2 mg/mL of bLF was dissolved in 0.1 M sodium carbonate buffer pH 9 and incubated overnight with FITC (Sigma-Aldrich, St. Louis, MO). Then, 50 mM NH4Cl was added for 2 hours at 4 °C to stop the reaction. The unbound FITC was eliminated by dialysis using cellulose membranes (Sigma-Aldrich, St. Louis, MO) at 4 °C.
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