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Partial selectivity can also be obtained by careful control of the reaction conditions. In another example, an amino acid having a chemical group that behaves as a nucleophile at a pH lower than neutrality, for example, aspartate or glutamate, can be a target amino acid and one or more other amino acids that behaves as a nucleophile at a pH less than neutrality can be a non-target amino acid that is not present in a labeled protein standard or modified in a labeled protein standard. For example, to test the consistency of migration between a labeled protein standard and its unlabeled counterpart, electrophoresis can be performed on a polyacrylamide gel, having a length of 8 cm, in which at the end of electrophoresis the dye front of the gel has migrated at least 5 cm, such as at least 6 cm, such as at least 6. Elution buffer: 8M urea, 200 mM Imidazole, 0. 50 ml centrifuge tubes. Blue Protein Standard, Broad Range, New England Biolabs. 5 ml of Column Conditioning solution (8M urea, 20 mM phosphate, 0.
In some illustrative embodiments, a selectively labeled protein standard selectively labeled on lysine is depleted in or lacks residues of at least one of cysteine, histidine, or tryptophan. 13 depicts the reaction scheme for generating the vinyl sulfone form of Orange 16. In some embodiments, a protein of a pre-labeled protein standard set that is selectively labeled on cysteine comprises an amino acid sequence derived from an nucleotide-disulfide oxidoreductase, such as a lipoamide dehydrogenase, a glutathione reductase, or a thioredoxin. The dye front can be a Coomassie dye front, such as a Coomassie G250 dye front. In exemplary embodiments, the selectively labeled protein lacks residues of a non-target amino acid capable of reacting with the dye. Fractions of 10 ml were collected and aliquots were run on a gel, and the purified protein fractions were pooled together. Optimal stability for up to 24 months. Labeling of Standard Proteins with Dyes. The standards can be labeled with two, three, four, or more visually distinguishable dyes. Non-synonymous amino acid alterations in PfEBA-175 modulate the merozoite ligand's ability to interact with host's Glycophorin A receptor. The pre-labeled protein standard set can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more selectively labeled proteins that comprises different numbers of copies of an amino acid sequence that is depleted in residues of a second amino acid. The column is washed extensively with Column Conditioning solution (8M urea, 20 mM phosphate, 0. Novex sharp prestained protein standard edition. Once the product was loaded onto the column the column was washed with 3 column volumes of water and then the product was eluted using 50% HPLC grade methanol in water. The 260 kDa protein had an estimated mass of 253, 624 daltons.
This generally occurs 14-17 hours after inoculation. In some illustrative embodiments, at least five, six, seven, eight, nine, or ten molecular weight markers can differ in size by increments that are multiples of 10 kDa. 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. In some preferred embodiments, the selectively labeled proteins having a molecular weight of greater than 10 kDa or greater do not differ by more than 5% in their migration in denaturing acrylamide electrophoresis gels from the migration of the same proteins in unlabeled form. As used herein, the term "protein" encompasses peptides. Novex sharp prestained protein standard.html. Synthesis of 50 kd PCR Inserts (1314 bp).
4 mM MgSO4; 220 μM dNTPs; and stabilizers; with the following primer sets: |50. This leads to a protein standard having variable label intensity per microgram of protein, and poor resolution of the protein standard in separation techniques that rely on mass, such as, but not limited to, electrophoresis and chromatography. Using the unique restriction site (Avr II), located between 50 kDa Thio repeat fragments 2 and 3 in the pTrc 160 kDa protein construct (FIG. The sample is run through the column and fractions are monitored using 280 nm detection. Malar J 19:367 (2020). Prestained protein ladder novex. 100 μl of 60 kDa BenchMark™ stock solution (OD=3. In particular, a protein that is "selectively labeled" on a [first] amino acid is a protein that has been conjugated with a labeling compound that has a reactive chemical group that is specific for the [first] amino acid, and that either has fewer than one residue per 10 kDa of one or more other (second) amino acids that can also react with the labeling compound, or has a chemical modification of one or more other (second) amino acids that can also react with the labeling compound. Otherwise the sample is warmed at 70° C. for 5 minutes to facilitate the solubilization of protein prior to centrifugation. Provided herein are labeled protein standards useful in electrophoresis or chromatography that have consistent separation characteristics that are substantially the same as the separation characteristics of their unlabeled counterparts. Proteins are covalently coupled with a blue chromophore except for two reference bands (one green and one red band at 25 kDa and 75 kDa respectively) when separated on SDS-PAGE(Tris-glycine buffer).
In some embodiments, a chromophore is a textile dye, such as for example, a Direct dye, a Disperse dye, a Dischargeable acid dye, a Kenanthol dye, a Kenamide dye, a Dyacid dye, a Kemtex reactive dye, a Kemtex acid dye, a Kemtex Easidye acid dye, a Remazol dye, a Kemazol dye, a Caledon dye, a Cassulfon dye, an Isolan dye, a Sirius dye, an Imperon dye, a phtalogen dye, a naphtol dye, a Levafix dye, a Procion dye, and an isothiocyanate dye. With the solution is stirring, sodium hydroxide was added dropwise to the stirred the solution until the pH is 10. 5 kDa to greater than 250 kDa. Molecular weight marker.
The solid dye was weighted and the yield was calculated. "Recombinant methods" is used interchangeably with "genetic engineering" and "recombinant [DNA] technology". PTrc 160 kd Expression Vector: TA clone 50. Any of the amino acids: cysteine, lysine, histidine, tryptophan, aspartate, glutamate, methionine, tyrosine, or asparagines can be target amino acids to which a labeling compound can be conjugated. The gels were run at 200 V until the dye front reached the bottom of the gel (6. For example, where lysine is a target amino acid to be conjugated with a dye, histidine and tryptophan, which are less reactive than lysine and cysteine but nonetheless can react with amino-reactive groups of labeling compounds, can optionally be considered non-target amino acids in addition to cysteine. Remaining liquid was removed, and the protein pellet was resolubilized in 50 mM Tris, 1% SDS pH=8 at high concentration (for example, 4 mg/ml or higher. ) A naturally-occurring protein can be any naturally-occurring protein. A dye used to label a selectively labeled protein standard of a pre-labeled protein standard set can be a fluorophore.
12 depicts a scheme for synthesizing 8-anilino-1-naphthalenesulfonic acid-aminophenyl vinyl sulfone (8-ANS-APVS). Not for use in diagnostic procedures. Preferably, a labeling compound used to label a protein standard has a high specificity for the reactive group of the target amino acid. The b-chain eluted in the wash buffer. A vector, protein-encoding sequences, etc. 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 BH6mer ORF was ligated into the digested pTrc vector backbone via BamHI-PmeI to generate the pTrc BH 60 kd expression construct having the insert shown in FIG. The proteins of a pre-labeled protein standard set provided in some preferred embodiments of aspects of the invention, when electrophoresed on a denaturing polyacrylamide gel, produce bands with widths that do not differ by more than two-fold between different proteins of the set that have molecular weights of 10 kDa or greater. 4-aminophenyl-2-sulfonatoethyl sulfone (2. Increasing or decreasing the number of target amino acid residues can be done to optimize the number of label molecules attached to a protein standard.
250 μl of 2 mg/ml 30 kDa (NL) stock solution was brought up to 1 ml volume to a final concentration of 50 mM Tris, 0. The expression clone was labeled pTrc1, 2, 3 Pme and renamed: pTrc 160 kd (FIG. The extracted trace was loaded in The baseline was adjusted and peaks were selected. 2A is a diagram of a nucleic acid construct (BH6mer ORF) having six copies of a truncated thioredoxin sequence lacking lysine separated by unique restriction sites. HIS purification is performed as follows: Toyopearl Chelate 650M resin (Tosoh Bioscience, Tokyo, Japan) is loaded with cobalt II chloride.
In some preferred embodiments, proteins standards are used in denaturing acrylamide gel electrophoresis in which proteins are denatured using a detergent, such as but not limited to SDS or LDS. D) incubating the protein with the reducing agent for a sufficient amount of time for cysteine-cysteine bonds to be reduced. One tablet of inhibitor is used for every 50 ml solution. The column is equilibrated with 50 mM Tris, 1% SDS pH=8. In preferred embodiments, each of the five or more labeled protein standards that has a molecular weight of 10 kDa or greater migrates within 5% of each of the five or more proteins in unlabeled form on the same acrylamide gels. 6 and the cells were incubated at 37° C. for an additional 4-6 hours. PCR colony screening identified 11/80 clones containing the LacZ insert and expression screening identified 5/11 clones having the LacZ insert in the correct orientation. 1D Gel Electrophoresis, Protein Gel Electrophoresis, Protein Gel Staining and Imaging, Proteins, Expression, Isolation and Analysis, Western Blotting. The column was washed with 8M urea in 50 mM Na-acetate pH=5.
In alternative embodiments, a selectively labeled protein that is depleted in a non-target amino acid can in some embodiments be a protein that comprises an amino acid sequence that has no known homology to a naturally-occurring protein, and can be designed and synthesized recombinantly or chemically, or using a combination of chemistry and recombinant technologies. At low pH the dye is a purple color and the fractions collected were in some cases checked by HPLC to assess purity. In some embodiments, a selectively labeled protein of the invention lacks residues of a second amino acid that can react with a labeling compound. Pre-labeled standards are labeled prior to separation or experimental procedures, and can be observed during or after separation procedures without performing additional steps required to stain the proteins in the midst of or at the conclusion of a separation or experimental procedure. A pre-labeled protein standard set can include one or more proteins that is not selectively labeled. In one aspect, the invention provides a pre-labeled protein standard set comprising a plurality of labeled proteins, in which one or more of the proteins of the plurality is selectively labeled, in which a selectively labeled protein comprises a labeling compound on a first, or target, amino acid, and has less than one residue of a second amino acid that reacts with the labeling compound per ten kilodaltons (kDa) of protein. The cells are harvested at early stationary phase, when two consecutive hourly readings of less than 0. These methods typically use standards for molecular weight or charge determination. The sample was then incubated for 10 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C. 50 μl of 1M iodoacetamide was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in the dark.