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Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome.
Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.
Document Type:

Reference

Product Catalog Number:

UFC901024
Product Catalog Name:

Amicon® Ultra-15 Centrifugal Filter Unit
Considerations when measuring urinary albumin: precision, substances that may interfere, and conditions for sample storage.
The measurement of small but abnormal amounts of albumin in urine is important in evaluating kidney disease in people with diabetes mellitus, hypertension, or possible adverse health effects from exposure to nephrotoxins. Routine laboratory methods for measuring albumin are not sensitive enough to measure the amounts that are significant in urine (less than 30 mg/L). In our laboratory we used three immunoassays for measuring urinary albumin: enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and immunoturbidimetric assay (IT). We calculated the CVs of the three methods, investigated potential interfering substances at three times their normal concentrations, and stored urine under different conditions to find the best way to protect the sample until assay. The potential interferents we checked were transferrin, urea, beta 2-microglobulin, retinol-binding protein, creatinine, kappa and lambda light chains, IgG, hemoglobin, ketone, and glucose. The stability study involved two study temperatures (-20 and -70 degrees C) and four treatments (centrifuging or filtering, before or after storage). We found the following: the RIA had the lowest CV; the results from the interference study showed no interference from normal physiological concentrations of the substances investigated; storage at -70 degrees C regardless of the treatment should be adequate to prevent loss of albumin immunoreactivity.
Document Type:

Reference

Product Catalog Number:

20-176
Product Catalog Name:

100X GTPγS, 10mM
Spinning-disk confocal microscopy of yeast.
Spinning-disk confocal microscopy is an imaging technique that combines the out-of-focus light rejection of confocal microscopy with the high sensitivity of wide-field microscopy. Because of its unique features, it is well suited to high-resolution imaging of yeast and other small cells. Elimination of out-of-focus light significantly improves the image contrast and signal-to-noise ratio, making it easier to resolve and quantitate small, dim structures in the cell. These features make spinning-disk confocal microscopy an excellent technique for studying protein localization and dynamics in yeast. In this review, I describe the rationale behind using spinning-disk confocal imaging for yeast, hardware considerations when assembling a spinning-disk confocal scope, and methods for strain preparation and imaging. In particular, I discuss choices of objective lens and camera, choice of fluorescent proteins for tagging yeast genes, and methods for sample preparation.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple