Ammonium acetate, triethylAmmonium acetate (Sigma, St. Louis, MO), and m-nitrobenzyl alcohol (Aldrich) were dissolved in HPLC grade water (Millipore, Burlington, MA) to concentrations of 100 mM. Bovine Cu/Zn superoxide dismutase (SOD) was obtained from MP Biomedicals (Santa Ana, CA), streptavidin (SA) from ProteoChem (Hurricane, UT), human hemoglobin (Hb) from Sigma Aldrich (St. Louis, MO), transthyretin (TTR) from Lee BioSolutions (Maryland Heights, MO), and C-reactive protein (CRP) from EMD Millipore (Burlington, MA). Each protein was diluted to a 50 μM stock of the protein monomer in 100 mM Ammonium acetate and passed through a size exclusion spin column (BioRad, Hercules, CA) for purification. Samples analyzed by nano-ESI were prepared as described previously by Wysocki and coworkers.16 Briefly, each protein was diluted to 4–10 μM monomer in three types of solutions: “standard” native solutions containing 100 mM Ammonium acetate; charge-reducing solutions containing 20 mM triethylAmmonium acetate (TEAA) and 80 mM Ammonium acetate; and supercharging solutions containing 20 mM m-nitrobenzyl alcohol (m-NBA) and 80 mm Ammonium acetate. All protein solutions had a final pH of approximately 7.
Human hemoglobin hb
Manufactured by Merck Group
Sourced in United States
Human hemoglobin (Hb) is a protein found in red blood cells that carries oxygen throughout the body. It is a crucial component of the blood and plays a vital role in the circulatory system. Hemoglobin consists of four globin subunits, each containing a heme group that can bind to oxygen molecules. This allows hemoglobin to efficiently transport oxygen from the lungs to the body's tissues.
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Lab products found in correlation
Ammonium acetate, by Merck Group (1 mentions)
M nitrobenzyl alcohol, by Merck Group (1 mentions)
Hplc grade water, by Merck Group (1 mentions)
Triethylammonium acetate, by Merck Group (1 mentions)
C reactive protein crp, by Merck Group (1 mentions)
Hplc grade ethanol, by Avantor (1 mentions)
Dichloromethane, by Avantor (1 mentions)
Ammonium persulfate, by Merck Group (1 mentions)
Fibrinogen from human plasma, by Merck Group (1 mentions)
Sodium phosphate monobasic, by Merck Group (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Cytochrome c cyt c, by Merck Group (1 mentions)
Artificial urine, by Avantor (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
6 protocols using human hemoglobin hb
1
Protein Analysis by Nano-ESI
2
Thiacarbocyanine Dyes and Biomacromolecules
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Thiacarbocyanine
(TC) dyes, 3,3′-diethylthiacyanine iodide
([TC0][I]), 3,3′-diethylthiacarbocyanine iodide ([TC1][I]),
and 3,3′-diethylthiadicarbocyanine iodide ([TC2][I]) were purchased
from Sigma Aldrich (St. Louis, MO). Lysozyme (Lys) from chicken egg
white, human transferrin (Trans) (>98%), albumin from human serum
(HSA) (approx. 99%), cytochrome c (Cyt-c), immunoglobulin G from human
serum (IgG), fibrinogen from human plasma (Fib), human hemoglobin
(Hb), ammonium persulfate, sodium phosphate dibasic, and sodium phosphate
monobasic were all also purchased from Sigma Aldrich. Lithium bis(trifluoromethane)sulfonamide
(Li[NTf2]) salt and lithium bis(pentafluoroethanesulfonyl)imide (Li[BETI])
salt were obtained from TCI Portland, Oregon. Artificial urine, HPLC-grade
ethanol, and dichloromethane were acquired from VWR (Batavia, IL).
Triple deionized ultrapure water (18.2 M Ω cm) was obtained
using an Aries high-purity water system (West Berlin, NJ). All reagents
were used as received without further purification.
(TC) dyes, 3,3′-diethylthiacyanine iodide
([TC0][I]), 3,3′-diethylthiacarbocyanine iodide ([TC1][I]),
and 3,3′-diethylthiadicarbocyanine iodide ([TC2][I]) were purchased
from Sigma Aldrich (St. Louis, MO). Lysozyme (Lys) from chicken egg
white, human transferrin (Trans) (>98%), albumin from human serum
(HSA) (approx. 99%), cytochrome c (Cyt-c), immunoglobulin G from human
serum (IgG), fibrinogen from human plasma (Fib), human hemoglobin
(Hb), ammonium persulfate, sodium phosphate dibasic, and sodium phosphate
monobasic were all also purchased from Sigma Aldrich. Lithium bis(trifluoromethane)sulfonamide
(Li[NTf2]) salt and lithium bis(pentafluoroethanesulfonyl)imide (Li[BETI])
salt were obtained from TCI Portland, Oregon. Artificial urine, HPLC-grade
ethanol, and dichloromethane were acquired from VWR (Batavia, IL).
Triple deionized ultrapure water (18.2 M Ω cm) was obtained
using an Aries high-purity water system (West Berlin, NJ). All reagents
were used as received without further purification.
3
Synthesis and Purification of Oligonucleotides
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Streptavidin, human hemoglobin (Hb), bovine serum albumin (BSA), SYBR Green I, Tween 20 and other reagents were purchased from Sigma-Aldrich (St. Louis, USA). Ni Sepharose 6 FF resin and His Mag Sepharose Ni magnetic beads (MB) were purchased from GE Healthcare (United Kingdom). “UltraMild” protected nucleoside β-cyanoethyl phosphoramidites and modified polymer supports for the synthesis of oligonucleotides were purchased from Glen Research (Sterling, USA). 2′-F-Modified pyrimidine triphosphates were purchased from Nanotech-C (Novosibirsk, Russia). Dulbecco's phosphate buffered saline (DPBS), T7 RNA polymerase, inorganic pyrophosphatase, DNase I, RevertAid reverse polymerase, FastAP thermosensitive alkaline phosphatase, T4 polynucleotide kinase and unmodified ribonucleoside triphosphates were purchased from Thermo Fisher Scientific (Waltham, USA). Taq DNA polymerase, and deoxyribonucleoside triphosphates were purchased from Biosan (Novosibirsk, Russia).
4
Growth Assay of S. aureus Strains
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S. aureus strains USA300 TCH1516 and USA300 FPR3757 were dilution plated similarly to previous description [69 (link)]. Cells were grown to stationary phase in TSB medium, and then diluted back to an OD600 of 0.05 upon which serial dilutions of 10−1 to 10−5 were made into fresh TSB medium. 5ul of each dilution was then plated in series onto either pre-warmed Tryptic Soy Agar (TSA), TSA II plates + 5% sheep’s blood (Fisher), or TSA plates supplemented with Human hemoglobin (Hb) (Sigma). Liquid cultures were allowed to dry on plates for up to an hour, and then plates were incubated at 37°C overnight. The plates were then imaged and the relative growth at each media condition was qualitatively assessed.
+ Expand
S. aureus strains USA300 TCH1516 and USA300 FPR3757 were dilution plated similarly to previous description [69 (link)]. Cells were grown to stationary phase in TSB medium, and then diluted back to an OD600 of 0.05 upon which serial dilutions of 10−1 to 10−5 were made into fresh TSB medium. 5ul of each dilution was then plated in series onto either pre-warmed Tryptic Soy Agar (TSA), TSA II plates + 5% sheep’s blood (Fisher), or TSA plates supplemented with Human hemoglobin (Hb) (Sigma). Liquid cultures were allowed to dry on plates for up to an hour, and then plates were incubated at 37°C overnight. The plates were then imaged and the relative growth at each media condition was qualitatively assessed.
5
Evaluating Solute Impact on Urine Specific Gravity
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Traditional CLSI-based interference testing was not applicable, as by definition all dissolved solutes contribute to SG depending on their concentration and molecular weight. Alternate experiments evaluating the extent of SG changes with increasing concentration of common urine solutes – albumin (bovine ALB; AMRESCO; Dallas, TX), Cr (Sigma Aldrich; St. Louis, MO), human hemoglobin (Hb; Sigma Aldrich), NaCl (BDH Chemicals, VWR; Radnor, PA), anhydrous d -glucose (VWR), and urea (Sigma Aldrich) - were therefore conducted. Bovine source ALB was chosen due to decreased cost and similar molecular weight (~66.5 kDa) to human albumin. Reconstituted human Hb was found to be in methemoglobin form [not detectable by Roche cobas 8000 H indices which use 600/570 nm wavelengths, but quantifiable by a COULTER Ac·T diff2 analyzer (Beckman Coulter; Brea, CA) using a cyanmethemoglobin method]. As hemolysate contains all RBC constituents (in addition to Hb) it was not appropriate for the present studies, as any additional solutes would also contribute to SG. Experiments were performed with serial dilutions (n=5) with solutes dissolved in a dilute urine pool matrix (baseline SG 1.0007).
6
Characterizing Hemoglobin-Sodium Dithionite Interactions
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Sodium dithionite (SDT) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Human hemoglobin (Hb) was obtained from Sigma-Aldrich (St. Louis, MO) and was employed as a model of HBOCs. Disodium hydrogen phosphate was purchased from Nacalai tesque (Kyoto, Japan). Sodium dihydrogen phosphate was purchased from Kanto Chemical Co., Inc., (Tokyo, Japan). One hundred mM phosphate buffer (pH 7.4) was prepared with Disodium hydrogen phosphate and Sodium dihydrogen phosphate. Hb (0.83 mg/mL) and SDT (0.16 -10 mg/mL) solutions were prepared with 100 mM phosphate buffer (pH 7.4).
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