The overloading of proteins of the standard set leads to bands on the gel that are broad and not sharply delineated, making it difficult to assess the migration distance of the protein of a particular molecular weight. Novex sharp prestained protein standard dual. 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. 5 kDa, such as at least about 5 kDa, or such as at least about 10 kDa. SDS PAGE protein ladder prestained.
The volume of the column was at least 20 times the volume of the sample for proteins labeled with the Red (8-ANS-APVS) dye. In some preferred embodiments, a pre-labeled protein standard set provided in a kit comprises at least five labeled proteins, in which two, three, four, or five of the labeled proteins are labeled on cysteine and lack lysine. 8; Imidazole; 5M HCl; Cobalt II chloride. Blue Protein Standard, Broad Range, New England Biolabs. In some embodiments of the method, the one or more selectively labeled protein standards The method includes applying the pre-labeled protein standard set to an electrophoresis gel, applying an electric field across the gel, and separating two or more protein standards of the pre-labeled protein standard set. 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. HIS purification is performed as follows: Toyopearl Chelate 650M resin (Tosoh Bioscience, Tokyo, Japan) is loaded with cobalt II chloride.
To our knowledge, customised protocols are not required for this product. The term "purified" as used herein refers to a preparation of a protein that is essentially free from contaminating proteins that normally would be present in association with the protein, e. g., in a cellular mixture or milieu in which the protein or complex is found endogenously such as serum proteins or cellular lysate. Please use the form below to provide feedback related to the content on this product. 50 1M Tris pH=8, 25 ul 20% SDS, and 800 μl ultrapure water were added to 125 μl of a 4 mg/ml solution of the 160 kDa (NL) standard protein. The protein solution plus TCA is incubated at 4° C. for 1-2 hours and then centrifuged at 8, 000×g for 10 minutes at 4° C. Novex sharp prestained protein standard chartered. The liquid is discarded and 30 ml of ultrapure H2O is added and mixed well. In embodiments in which the protein standard is made using recombinant methods, one or more mutations can be introduced into the nucleic acid sequence encoding the standard protein, where at least one mutation can alter a codon to change the number of residues of a target amino acid, or the position of a target amino acid.
Additional target amino acid codons can be added to a nucleic acid sequence that encodes a protein standard of the invention. 1 forward primer (SEQ ID NO:20) and 50. In another aspect, the invention provides methods of providing a set of pre-labeled protein standards to a customer, in which the set of pre-labeled protein standards includes any of the pre-labeled standard sets and kits disclosed herein. Ab116028 has been referenced in 16 publications. The invention includes in some illustrative embodiments a set of pre-labeled protein standards that includes at least two proteins of different molecular weight that are labeled on cysteine and lack lysine residues. Novex sharp prestained protein standard version. 5 kDa (such as, for example, having a molecular weight of greater than 5 kDa, such as, for example, having a molecular weight of 10 kDa or greater) have substantially the same migration on electrophoresis gels as their unlabeled counterparts.
In some preferred embodiments, the widths of visually detectable bands produced by at least five pre-labeled proteins of a standard set do not differ by more than 30%. 5 cm apart at the completion of electrophoresis. CCGGCGGCCGATGTGTGATCGTATTATTCAT, |50. In this case protein sequences can optionally be selected base on the abundance of cysteine and the paucity of lysine in the amino acid sequence used, which in some embodiments can reduce the number of codons to be mutated. The Fisher Scientific Encompass Program offers items which are not part of our distribution portfolio. "Homologous" means that a protein peptide, or amino acid sequence has at least 65%, at least 70% amino acid sequence identity, at least 80% amino acid sequence identity, preferably 90% amino acid sequence identity, and more preferably at least 95% amino acid sequence identity with amino acid sequence referred to. In a further aspect, the invention provides methods of labeling proteins that include attaching a label to one or more cysteine residues to a protein that lacks lysine residues.
The PCR inserts were TA cloned into pCR2. The proteins of a pre-labeled protein standard set provided in a kit preferably span a molecular weight range of from 10 kDa or less to 100 kDa or more, and can span a molecular weight range of from 5 kDa or less to 250 kDa or more. In some embodiments, one or more codons of the second amino acids is deleted from the nucleic acid sequence to delete amino acid residues from a standard protein that are capable of reacting with a labeling compound. The solution became clear and was cooled to room temperature.
The standards can be labeled with two, three, four, or more visually distinguishable dyes. Such variability in the population of labeled protein results in a range of masses for the particular labeled protein, depending on the range in the amount of dye molecules attached to the protein. In some aspects of the invention, a pre-labeled protein standard set can include one or more copies of an amino acid sequence having at least 70% or at least 80% identity to at least 20, at least 30, at least 40, or at least 50 contiguous amino acids of a naturally-occurring protein in which the amino acid sequence comprises one or more amino acid changes that alter the number or spacing of a first amino acid targeted for labeling. 5 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to about 80 kDa are at least 3. 8 cm from the bottom of the sample wells). The ligation reaction was transformed into One Shot® Top 10 competent bacterial cells (Invitrogen, Carlsbad Calif., USA) and the resulting colonies were PCR screened for the LacZ gene. Prism protein ladder. For example, labeling of a particular protein with a dye that has high specificity for a first amino acid and reduced specificity for a second amino acid can result in a population of labeled protein variants, in which the variants are predominantly labeled on the first amino acid, but vary in the degree of labeling of the second amino acid that is present on the protein. The width of bands visible to the naked eye from proteins having a molecular weight of greater than 3. Two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more copies of the nucleic acid sequence encoding a truncated thioredoxin can be assembled together to make a recombinant protein having multiple copies of a truncated thioredoxin sequence. The method used for purification was the following: insulin was solubilized at 5 mg/ml in 8M urea, 50 mM Tris pH=8.
The resin-bound dye was then washed to remove most of the acid from the coupling step. 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. In embodiments in which at least one of lysine, histidine, or tryptophan is a target amino acid, a label preferably includes an amino-reactive group for conjugation to the standard. The standards can span a molecular weight range of from less than 10 kDa to greater than 100 kDa, or from less than 5 kDa to greater than 250 kDa. 5 kDa migrate within 5% of the migration distance of the same proteins that are not labeled. Many denaturing polyacrylamide gel electrophoresis systems are known in the art, such as, for example, Bis-Tris gels, Tris-tricine gels, Tris-acetate gels, or Tris-glycine gels. The invention additionally provides sets of pre-labeled protein standards that can be used as molecular weight markers in biochemical separations, in which at least one labeled protein of the sets is selectively labeled on a first amino acid. This clone was subsequently designated pTrc 260 kDa (FIG. In making labeled protein standards of the invention, a target amino acid is an amino acid whose labeling is intended; the labeling of a protein on a target amino acid is achieved by selecting a labeling compound with a reactive chemical group that reacts with the reactive chemical group on the target amino acid. The method can also include staining the unlabeled protein prior to detecting the unlabeled protein. The cells are harvested at early stationary phase, when two consecutive hourly readings of less than 0. 14B shows the same set of markers in unlabeled form electrophoresed on a 4-12% Bis-Tris gel with MES running buffer. Use at an assay dependent concentration. 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 expression clone was labeled pTrc1, 2, 3 Pme and renamed: pTrc 160 kd (FIG. The gel purified vector was ligated with TA clone 50. 217: 220-230; and Schagger H (2001) Methods Cell Biol. 5 μl of 4×LDS and 2 μl NuPAGE reducing reagent were added to 15 μl of the whole lysate and to 15 μl of insoluble fraction. In these embodiments, the two, three, four, or five labeled proteins can have between two and seven, or between two and five, cysteine residues per 10 kDa. The columns were washed with 50 mM Tris, 0. Extracting the protein is performed as follows: 10 ml BugBuster® HT protein extraction reagent (Novagen, Madison, Wis., USA) with Complete Protease Inhibitor (Roche Applied Science, Indianapolis, Ind., USA) is added per every 1 g cell paste. The samples were incubated for 10 minutes at 70° C. 10 μl of each sample were loaded on a 4-12% NuPAGE® gel and run with 1×MES running buffer at 200V for 37 minutes. In some preferred embodiments, an amino acid sequence is derived from a thioredoxin sequence, having at least 70% or at least 80% identity with the amino acid sequence of at least 20, at least 30, at least 40 or at least 50 amino acids of a thioredoxin, such as a truncated thioredoxin.
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