After 4 h of BD, the lung-heart block was retrieved, the pulmonary artery was cannulated and the left ventricle excised for drainage. After flush it was preserved in cold Perfadex® (XVIVO Perfusion, Sweden) for 1 h (cold storage). The normothermic EVLP method (4 h) was adapted from the study by van Zanden et al. [19 (link)]. After placing the lung-heart block in the closed system, the recruitment maneuver was performed for 3 s [15 cmH2O of PEEP, and 20 cmH2O of peak inspiratory pressure (PIP)]. Mechanical ventilation (Babylog 8000 ventilator, Draeger, Germany) was set with a tidal volume of 7 mL/kg body weight, PEEP of 5 cmH2O, frequency of 60 breaths/min, and fraction of inspired oxygen (FiO2) set at 21%. For perfusion, we used Perfadex® solution (XVIVO Perfusion, Sweden), supplemented with albumin (GE Healthcare, Austria) and augmentin (Sandoz, The Netherlands) (homemade STEEN solution). The lungs were gradually rewarmed to 37 °C (1 °C/ 2 min). Perfusion started with a pulmonary arterial pressure (PAP) of 9 mmHg, and after 10 min, maintained at 12 mmHg. The ventilation and flow parameters were continuously recorded. Samples were collected from the perfusate leaving the lung, after the stabilization period, at 15 and 240 min for analysis.
Albumin
Manufactured by GE Healthcare
Sourced in Austria, China, United Kingdom, Spain
Albumin is a laboratory product that serves as a standard solution for the measurement of total protein levels in biological samples. It is a purified protein derived from human serum, which is used to calibrate and validate testing equipment to ensure accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Thyroglobulin, by GE Healthcare (5 mentions)
Ferritin, by GE Healthcare (4 mentions)
Aldolase, by GE Healthcare (4 mentions)
Blue dextran, by GE Healthcare (4 mentions)
Alcohol dehydrogenase, by GE Healthcare (3 mentions)
Albumin, by Merck Group (3 mentions)
Ovalbumin (ova), by GE Healthcare (3 mentions)
Carbonic anhydrase, by GE Healthcare (2 mentions)
Ribonuclease, by GE Healthcare (2 mentions)
Catalase, by GE Healthcare (2 mentions)
Bovine rnase a, by GE Healthcare (2 mentions)
Akta fplc, by GE Healthcare (2 mentions)
Sephadex g 10, by GE Healthcare (2 mentions)
Rnase, by GE Healthcare (1 mentions)
Hiload 16 600 superdex 200, by GE Healthcare (1 mentions)
Millex ha, by Merck Group (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
Igg depletion spintrap columns, by GE Healthcare (1 mentions)
Lysc endoproteinase ms grade, by Thermo Fisher Scientific (1 mentions)
14 protocols using albumin
1
Normothermic Ex Vivo Lung Perfusion
2
Analyzing Protein Polymerization States
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gel filtration with the FPLC system can be also employed to analyze the polymerization state of native or active proteins26 (link). Protein samples were injected into the column for separation, which was equilibrated with the buffer (20 mM Tris–HCl, 150 mM NaCl, 10% glycerol, pH 7.5). Five molecular weight markers were used: beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa) (GE Healthcare, China agency). Protein separation was monitored by measuring the absorbance at 280 nm. To analyze the effects of acidic or more alkaline conditions on the protein polymerization state, the above-mentioned buffer was adjusted to pH 5.5 and 8.5, respectively.
3
Degreased HTDDRB Rice Powder Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HTDDRB (130 °C, 30 min after moist heat stabilization treatment, the powder is degreased) was purchased from Heilongjiang Beidahuang Rice Industry Group Co., Ltd. (Harbin, China); 5,5′-disulfide (2-nitrobenzoic acid) (DNTB), guanidine hydrochloride, sodium dodecyl sulfate (SDS), β-mercaptoethanol (Shanghai Boao Biotechnology Co., Ltd., Shanghai, China), the above are biotechnology grade.8-aniline-1-naphthalene sulfonic acid (ANS) (Amresco Co., Ltd., Solon, OH, USA); Sodium dihydrogen phosphate, disodium hydrogen phosphate, urea, ethylenediamine tetraacetic acid (EDTA) (Tianjin Bodi Chemical Co., Ltd., Tianjin, China), all the above reagents are analytically pure. High relative molecular weight standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (75 kDa), ooalbumin (44 kDa), ribonuclease (13.7 kDa), (GE-Healthcare UK Limited).
4
Size Exclusion Chromatography of C-LrtA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SEC experiments were performed at pH 8.0 (50 mM Tris) and 0.250 M NaCl in a Superose 12 10/300 GL column, which was connected to an AKTA FPLC (GE Healthcare, Barcelona, Spain); absorbance at 280 nm was monitored during the elution. Flow rate was 0.8 mL/min, and C-LrtA was loaded in a volume of 100 μL at several protein concentrations, in the range of 30–500 μM (in protomer concentration). The markers used to calibrate the column were ferritin, catalase, aldolase, albumin, bovine RNase A and blue dextran (GE Healthcare, Barcelona, Spain); the isolated protein markers were loaded in the column in the above described buffer, at the same flow rate, and three independent measurements were performed with each marker.
5
Radiolabeling of Albumin with Technetium-99m
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following established procedures50 (link), albumin (Sigma, St. Louis MO) was labeled with 99mTc (GE Healthcare, Piscataway, NJ). A mixture of 240 mg/ml stannous tartrate and 1 mg/ml albumin was adjusted to pH 3.0 with HCl. One millicurie of 99mTc-NaOH4 was added to this mixture and allowed to incubate for 20 min. The 99mTc-albumin was purified on a column of G-10 Sephadex (GE Healthcare) in 0.1 ml fractions of phosphate buffer (0.25 M). Radioactivity in the purified 99mTc-albumin peak was more than 90% acid precipitable in an equal volume of 1% bovine serum albumin (BSA) and trichloroacetic acid (30%). 5 × 106 cpm/mouse of purified 99mTc-albumin fraction was prepared in a final volume (0.2 ml/mouse) of lactated Ringer’s solution containing 1% BSA.
6
Fractionation of HEK293T cell extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were incubated with Roeder A buffer (22 (link)) in an appropriate amount for 10 min at room temperature, dounced 10 times, and adjusted to 150 mM NaCl. After centrifugation at 17 000 g for 30 min the supernatants (S100 extracts) were filtrated with Millex-HA, 0.45 μm filter unit (Merck Millipore) and applied to a Superdex 200 HiLoad 16/600 or Superdex 200 increase 10/300 GL column (GE Healthcare). 2 ml respectively 0.5 ml fractions were collected in running buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.5) and analyzed by immunoblotting. The columns were calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and RNase (14 kDa) (GE Healthcare).
7
Fractionation of Whole Cell Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell extracts (200 μl containing 500 to 1,000 μg of protein) of control or heat-shocked cells were applied on a Superdex 200 10/300 GL chromatographic separation column with an AKTA fast protein liquid chromatography apparatus (GE Healthcare). The samples were eluted at 0.3 ml/min with a buffer containing 1% glycerol, 20 mM Tris-HCl (pH 7.9), 200 mM KCl, and 1.5 mM MgCl2. The fractions (0.5 ml) were precipitated with trichloroacetic acid (10%, final concentration), washed with acetone, dried, suspended in gel sample buffer, and analyzed by SDS-PAGE and Western blotting. The protein standards were as follows: thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; albumin, 67 kDa (GE Healthcare).
8
Targeted Protein Quantification Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Albumin and IgG Depletion SpinTrap columns were purchased from GE Healthcare (cat.no. 28-9480-20). Sequencing grade modified trypsin was obtained from Promega (cat.no. V5111) and LysC endoproteinase MS grade was purchased from Thermo Scientific (cat.no. 90051). Solid phase extraction cartridges, Sep Pak tC18, 50 mg, were obtained from Waters (cat.no.WAT054960). SIL peptides were synthetized with a heavy C terminal lysine or arginine (C terminal arginine, 13C6, 15N4, Δm = 10 Da, C terminal lysine 13C6, 15N2, Δm = 8 Da) or when it was not applicable with a heavy leucine 13C6, 15N1, Δm = 7 Da or phenylalanine 13C9, 15N1, Δm = 10 Da. (Thermo Fisher, crude quality). The synthetic peptides were mixed together from the stock solutions (50% acetonitrile, 0.1% TFA), aliquoted in Eppendorf Low Bind tubes and stored at 80°C before single use. All other reagents were obtained from Sigma-Aldrich.
9
Molecular Weight Estimation of Bp-13 PLA2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tricine SDS-PAGE in a discontinuous gel and buffer system was used to estimate the molecular mass of Bp-13 PLA2, under reducing and nonreducing conditions [20 (link)]. The used molecular weight markers in kDa were phosphorylase B: ~ 94, albumin: 67, ovalbumin: 43, carbonic anhydrase: 30, soybean trypsin inhibitor: 20, and α-lactoalbumin: 14 (GE Healthcare).
10
Radiolabeled Albumin and Sucrose Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following established procedures39 (link), albumin (Sigma, St. Louis MO) was labeled with 99mTc (GE Healthcare, Piscataway, NJ). A mixture of 240 mg/ml stannous tartrate and 1 mg/ml albumin was adjusted to pH 3.0 with HCl. One millicurie of 99mTc-NaOH4 was added to this mixture and allowed to incubate for 20 min. The 99mTc-albumin was purified on a column of G-10 Sephadex (GE Healthcare) in 0.1 ml fractions of phosphate buffer (0.25 M). Radioactivity in the purified 99mTc-albumin peak was more than 90% acid precipitable in an equal volume of 1% bovine serum albumin (BSA) and trichloroacetic acid (30%). 5 × 106 cpm/mouse of purified 99mTc-albumin fraction was combined with 1 × 107 dpm/mouse of 14C-sucrose (Perkin Elmer, Waltman, MA) in a final volume (0.2 ml/mouse) of lactated Ringer’s solution containing 1% BSA.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!