In some preferred embodiments in which a first amino acid is cysteine, and the reactive group of cysteine is a sulfhydryl group, the method preferably also comprises: - c) prior to a), combining a protein that comprises one or more cysteine residues with a reducing agent; and. The invention includes pre-labeled protein standard sets that comprise a plurality of labeled proteins, in which one or more of the labeled proteins is depleted in lysine residues and comprises a labeling compound conjugated to one or more cysteine residues. ACTCTGCCCAGAAGTCGAC. Highly Resolving Electrophoretic Separation of Pre-Labeled Protein Standards. Nucleotide-disulfide oxidoreductases are highly soluble proteins (an advantage for accessibility of residues for labeling) having an abundance of cysteine residues. In another example, glutamate, aspartate, and the C-terminal amino acid of a protein can be target amino acids, where a dye conjugated to the selectively labeled protein includes a reactive chemical group that reacts with carboxylates.
The expression clone was labeled pTrc1, 2, 3 Pme and renamed: pTrc 160 kd (FIG. 199: 223-231; Schagger H, Cramer W A, and von Jagow G (1994) Anal. As used herein an amino acid or reactive group of an amino acid that "reacts with" a labeling compound becomes covalently bound to the labeling compound. The present invention seeks to reduce labeling of non-target amino acids by reducing their occurrence in a protein used as a pre-labeled protein standard. The cell paste is vortexed for 10-20 seconds to break the pellet and the paste is mixed with the Polytron right away. 3A shows the map of the pTrc BH 60 kDa cloning construct used to generate the lower molecular weight pTrc BH 30 kDa construct (shown in FIG.
Effects of chemotherapy on placental development and function using in vitro culture of human primary cytotrophoblasts. The incubation can occur at any temperature, from close to 0 degrees C. to about 90 degrees C., but typically is for about 1 hour at room temperature or above (such as up to 60 degrees C. ) to several hours on ice. The term "reactive group" or "reactive chemical group" as used herein refers to a chemical group that is capable of reacting with another chemical group to form a covalent bond, i. e. is covalently reactive under suitable reaction conditions, and generally represents a point of attachment for another substance. In some embodiments of this aspect of the invention, a selectively labeled protein includes an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein, in which the naturally-occurring protein is naturally depleted in or deficient in a non-target amino acid. 14A shows a pre-labeled protein standard set of the invention electrophoresed on a 4-12% Bis-Tris gel with 1×MES running buffer. The solution was then cooled back to 0° C. to precipitate the diazonium salt. By reducing the number of residues of amino acids that can bind a labeling compound in side reactions, variability in the amount of labeling compound attached to a given protein molecule is reduced. A "nontarget amino acid" can have the same reactive chemical group as a target amino acid or a different reactive chemical group. A negative ion mode mass spectrum was obtained to be sure that a parent peak was seen at a mass to charge ratio of 492. The samples were analyzed for migration on 8 cm×8 cm 4-12% BisTris/MES gels, 4-12% BisTris/MOPS gels, and 4-20% Tris Glycine gels. Reagents: Complete Protease Inhibitor (Roche Applied Science, Indianapolis, Ind., USA); Freshly prepared 25 mg/ml lysozyme (Calbiochem, San Diego, Calif., USA) in ultrapure water; Induced cell culture as for 30, 40, 50 and 110 kDa (NL) proteins; Amberlite MB-150 (Sigma-Aldrich); Toyopearl AF Chelate 650M (Tosoh Bioscience, Tokyo, Japan); CHAPS detergent; Urea; 1M Na-phosphate pH=7.
Fractions of 10 ml were collected and aliquots were run on a gel, and the purified protein fractions were pooled together. De-ionize for 2 or more hours with 10 g/liter Amberlite mixed bed resin. The method includes: reducing cysteines of a protein that lacks lysine residues and adding a labeling compound to the protein under conditions that allow conjugation of the dye with cysteine. Similarly, "about 100 mM" (or "approximately 100 mM") encompasses a range of concentrations from 90 mM to 110 mM, inclusive. In some aspects, the invention includes a method for making a protein standard, comprising attaching a label to one or more lysine residues of a proteins that is depleted in cysteine residues. Electrophoretic Migration. To generate chimeric nucleic acid molecules, generate nucleotide sequence changes, or add or delete nucleic acids to a nucleic acid sequence.
The modified pTrc LacZ-Flash vector was digested with BamHI-Not I and the gel purified (4377 bp) vector was ligated with the TA 50. Supplier Catalog Number:||JB-EPL-2500|. 8 L non-baffled seed flask of approximately 1 liter of rich media with a freshly transformed (less than one week old) colony containing the expression plasmid. Using the pTrc BH 60 kDa expression construct of Example 1 as the PCR template, several 50 kDa inserts were generated using Platinum® PCR Supermix High Fidelity PCR mix (Invitrogen; Carlsbad, Calif. ) that contained Taq DNA polymerase, Pyrococcus species GB-D thermostable polymerase, Platinum® anti-Taq polymerase antibody, 66 mM Tris-504 (pH 8. The gel was then scanned at 300/300 dpi and saved as gray scale '' image. Population genetic and biophysical evidences reveal that purifying selection shapes the genetic landscape of Plasmodium falciparum RH ligands in Chhattisgarh and West Bengal, India. The 20 kDa BenchMark™ protein standard includes a truncated thioredoxin fragment fused to two copies of a 5 kDa fragment of the E. coli DEAD-box protein (as disclosed in U. Comprehensive - a wide range of molecular weight bands consisting of 12 proteins in the range of 3. 5%, within 2%, within 1. The dye fractions were combined and the solvent was removed in vacuo using a rotary evaporator.
In some preferred embodiments, the two or more labeled proteins are selectively labeled on a first amino acid and comprise one or more copies of an amino acid sequence of a naturally-occurring protein or having at least 70% or at least 80% identical to at least 20, at least 30, at least 40, or at least 50 contiguous amino acids of a naturally-occurring protein. This clone, labeled pTrc 50. The sample is allowed to cool down for 5 minutes at room temperature (or until the temperature drops to 30° C. ) and then 5. In some embodiments, a pre-labeled protein standard set includes at least ten labeled proteins spanning a molecular weight range of from 10 kDa or less to 250 kDa or greater, in which the electrophoretic migration of 70% of the labeled protein standards having a molecular weight of 10 kDa or greater is within 2% of the electrophoretic migration of each of the protein standards in unlabeled form. 8 kDa, so that the labeling compounds do not substantially alter separation rates of the proteins in electrophoresis or chromatography, for example. CACACAGGAAACAGCTATGA. 6A shows a map of the pTrc BH 50 kDa "No Lysine" construct. 5 residues of cysteine, per 10 kDa. 14B shows the same set of markers in unlabeled form electrophoresed on a 4-12% Bis-Tris gel with MES running buffer. The fractions with the purified proteins are pooled together and the pH is adjusted to 7.
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