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Dialyzable and Nondialyzable Transfer Factor
Published in Edward P. Cohen, A. Arthur Gottlieb, Immune RNA, 2020
A number of investigators have also reported on the fractionation of human and nonhuman primate TFd using either Sephadex G-25 or G-10 or Bio-Gel.10,11,31,59,67-69 Although there are slight differences in the chromatograms obtained by various investigators, these are in part due to the eluent used. Ammonium bicarbonate and phosphate buffers have been used most frequently, but adequate separation is also obtained when distilled water is used as the eluent. A number of investigators are in agreement that the peaks which transfer DH have an unusually high 260:280 absorbance ratio which may in part be due to substances other than polyribonucleotides.
Urologic procedures
Published in J. Richard Smith, Giuseppe Del Priore, Robert L. Coleman, John M. Monaghan, An Atlas of Gynecologic Oncology, 2018
Padraic O’Malley, Peter N. Schlegel
Beyond the possible metabolic acidosis, urinary diversion can be associated with a number of other metabolically related disorders including vitamin B12 deficiency and osteomalacia. The most common segment utilized for diversion is the terminal ileum. Absorption of vitamin B12 occurs primarily at this point. The rates of vitamin B12 are unknown among patients with urinary diversion, although some have reported they can be as high as 30% (Pfitzenmaier et al. 2003). Usually development of the deficiency requires 3 to 5 years after surgery for the body’s stores to have become depleted. However, serious neurological sequelae can occur as a result. Further neurological sequelae also occur as a result of magnesium deficiency, drug intoxication, and abnormalities of ammonium/bicarbonate metabolism in patients with urinary diversion. A clinician needs to keep these in mind in the long-term follow-up of their patients and be vigilant for signs of any of these metabolic derangements.
Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
The urinary light chains from patients were purified (1) using dialysis, Affi-Gel blue treatment and gel filtration chromatography. SDS-PAGE and Western blot analyses were performed to determine which fractions contained the light chain. Aliquots from samples were treated with Asp-N, Glu-C, Lys-C, or trypsin in ammonium bicarbonate buffer. The enzymatic digests were analyzed using a Waters CapLC capillary HPLC coupled with an Applied Biosystems QSTAR Pulsar i quadrupole/orthogonal acceleration TOF mass spectrometer. A Magic Michrom C18 column (0.320 x 150 mm) was used for the separation at a flow rate of 1 uL/min (500 nL/min into the MS). The gradient was 5-50% B in 50 min followed by 50-70% B in 15 min (buffers A: 0.1% formic acid in water, buffer B: 85% acetonitrile 10% isopropanol and 5% water with 0.1% formic acid). The mass spectrometer was run in the IDA (information-dependent acquisition) mode with rolling collision energies, enabling the automated acquisition of MS/MS data from selected precursor peptide ions.
First trimester serum apolipoproteins in the prediction of late-onset preeclampsia
Published in Scandinavian Journal of Clinical and Laboratory Investigation, 2023
Emma J. Bendix, Julie D. Ravn, Lene Sperling, Martin Overgaard
Biomarker analysis was performed using a multiplex multiple-reaction-monitoring mass spectrometry (MRM-MS) assay for serum apolipoproteins (Apo) A-I, A-II, A-IV, B-100, C-I, C-II, C-III, D, E, H, J, and M. Serum samples from cases and controls were subjected to MRM-MS, thus pre-specified peptides were quantified, by normalization of their MS peak area of selected precursor/product ion pairs to that of the corresponding spiked-in heavy isotope labeled peptides. The following is a summary: 10 µL of diluted serum, 1:20 in 50 mM ammonium bicarbonate, was denatured, reduced, alkylated and trypsinized essentially as described by Ravnsborg et al. [23]. Individually adjusted amounts of 41 heavy isotopes labeled standard peptides SpikeTidesTM_L (JPT Peptide technologies, Berlin, Germany), were added to each sample in approximation of a 1:1 ratio to the endogenous light peptides. Peptides were purified on EvoTips and separated online on a 150 µm ID, 8 cm analytical column using an Evosep One/TSQ Altis LC-MS system interfaced with an EASY-Spray ion source (Evosep Biosystems and Thermo Fisher Scientific). Samples were handled and analyzed batch-wise in 96-well format using a fixed 60 samples/day LC program according to the manufacturer mode. Coefficient of variation (CV) calculations (intra- and inter-assay) were based on four times analyses of a serum pool for each batch. The serum samples were divided and analyzed in three batches. Intra-assay CV’s % were in the range 2–8%, 6–10% and 10–37% for the three batches, while the mean inter-assay CV was 12%.
Evaluation of molecular brain changes associated with environmental stress in rodent models compared to human major depressive disorder: A proteomic systems approach
Published in The World Journal of Biological Psychiatry, 2018
David Alan Cox, Michael Gerd Gottschalk, Viktoria Stelzhammer, Hendrik Wesseling, Jason David Cooper, Sabine Bahn
MDD tissue storage, preparation and proteomic abundance comparisons were performed as previously defined (Gottschalk et al. 2014). Approximately 12–16 mg of mouse tissue per sample were used for the SD model and 22–28 mg of rat tissue per sample were used for the CMS and PNS models. A previously published protein tissue isolation protocol was applied to all rodent model tissue samples (Ernst et al. 2012). Samples were added to a fractionation buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% ASB14 and 70 mM DTT at a 5:1 (v/w) ratio (Martins-de-Souza et al. 2007). Sonification and vortexing (at 4 °C for 30 min) of the samples was carried out before centrifuging at 17,000 × g at 4 °C. A Bradford assay (Bio-Rad) was used to determine protein concentrations of the supernatants in triplicate, using acetone to precipitate proteins (approximately 100 μg) from each sample. One hundred microlitres of ammonium bicarbonate (50 mM) were used to dissolve the precipitates, before protein concentrations were determined. Reduction of protein sulfhydryl groups was carried out using 40 μg of proteins and 5 mM DTT at 60 °C for 30 min. Alkylation was carried out using 10 mM iodoacetamide and incubating in the dark at 37 °C for 30 min. Protein digestion was performed using porcine tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin at a 1:50 (w/v) ratio for 17 h at 37 °C. Reactions were stopped via the addition of 8.8 M HCl at a 1:60 (w/w) ratio.
Site-specific N- and O-glycosylation analysis of atacicept
Published in mAbs, 2019
Kathrin Stavenhagen, Rabah Gahoual, Elena Dominguez Vega, Angelo Palmese, Agnes L. Hipgrave Ederveen, Francesca Cutillo, Wolf Palinsky, Horst Bierau, Manfred Wuhrer
In-gel treatment with PNGase F was performed in triplicates similar to the in-gel protease treatment with some modifications. Twenty-five mM ammonium bicarbonate was replaced during the whole procedure by 25 mM sodium bicarbonate. For PNGase F treatment, 30 µl digestion buffer, containing 2U PNGase F in 2% NP-40/1 x PBS, were added to the dry gel pieces and incubated on ice for 45 min to swell the gel pieces, before incubation at 37°C overnight. Next, the supernatant was removed and another 20 µl of water was added and incubated for 30 min in a sonication bath. The supernatant was again removed, combined with the first one and stored at −20°C before use.