Up to 100% electroblot transfer efficiency (Seema Qamar, CIMR, Cambridge University 2018). 5%, or 1% of one another are selectively labeled on a first amino acid. Elite Pre-stained Protein Ladder vs Novex Sharp Pre-stained Protein Standard (ThermoFisher). 50 μl of the lysate was transferred to a separate tube. 21, 2007 and having a size of 87. 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. These products typically do not have pictures or detailed descriptions. Unambiguous - each band in the standard is pre-stained with a unique color for easy interpretation of results. In some embodiments of these aspects, one, two, three, four, five, or more than five labeled proteins of a protein standard set having molecular weights of 10 kDa or more are selectively labeled on a target amino acid and migrate substantially the same as their unlabeled counterparts. Novex sharp prestained protein ladder. A dye can be, for example, a chromophore or a fluorophore.
Lysozyme was used as a 15 kDa molecular weight marker. The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user. 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. The liquid is discarded and 30 ml of ultrapure H2O is added and mixed well. In some preferred aspects, the present invention provides protein molecular weight standards that are selectively labeled, such that attachment of a dye to an amino acid that is not targeted for labeling (a non-target amino acid) is restricted. Novex sharp prestained protein standard curve. 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. 5 kDa to greater than 250 kDa. Selectivity of labeling is best obtained by selection of an appropriate reactive dye. 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 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.
16 mm, a difference of just under 2-fold. Mutation of a codon can be to any codon for an amino acid other than the non-target amino acid. Proteins made by recombinant methods can be based on the sequences of naturally-occurring proteins, or can have synthetically designed sequences. 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. Novex sharp prestained protein standard dual. In one aspect, the invention includes a pre-labeled protein standard set that includes two or more proteins selectively labeled on a first amino acid with a labeling compound and depleted in a second amino acid capable of reacting with the labeling compound, in which the two or more selectively labeled proteins includes different numbers of copies of an amino acid sequence having at least 70% homology to at least 30 contiguous amino acids of a sequence of a naturally-occurring protein. Protein expression screens in BL21 DE3 STAR were preformed to validate PCR screen screening results. In some preferred embodiments, a target amino acid of a pre-labeled protein standard can be an amino acid such as, but not limited to, cysteine, lysine, histidine, tryptophan, aspartic acid, glutamic acid, tyrosine, arginine, methionine, an N-terminal amino acid of the protein, or a C-terminal of the protein, in which one or more amino acids that also can undergo nucleophilic addition are non-target amino acid(s) that can be depleted in a pre-labeled protein standard.
Preferably, the calculated molecular weights for a pre-labeled protein standard having a molecular weight greater than 5 kDa and its unlabeled counterpart on one of the referenced denaturing acrylamide gels are within 10%, 7%, or 5% of one another. The dye front can be a Coomassie dye front, such as a Coomassie G250 dye front. In a separate 50 mL flask, 0. Category:||Molekularbiologie|. 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. 4-aminophenyl-2-sulfonatoethyl sulfone (2.
25 lpm air, 500 rpm agitation, and the pH is controlled to 6. 5 mm) or larger gel. Dyes can include reactive groups, such as cysteine reactive groups (e. g., maleimide, iodoacetic acid, iodoacetamide, and vinyl sulfone) or amino reactive groups (such as, for example, isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NETS) esters, sulfonyl chlorides, aldehydes, ketones, glyoxals, epoxides, oxiranes, carbonaes, aryl halides, imidoesters, carbodiimides, and acid anhydrides). The program measured the width of the bands where the intensity of the image was 50% or more of the maximum intensity peak height for (FIG. In some embodiments, the protein that is depleted in cysteine residues comprises an amino acid sequence that has homology to at least 40 amino acids of a naturally-occurring protein, such as at least 70%, at least 80%, or at least 90% homology to at least 40 amino acids of a naturally-occurring protein, and has fewer cysteine residues than the amino acid sequence of the naturally-occurring protein to which has homology. The sequence-verified Thio repeat ORF insert (BH6mer ORF) from BlueHeron® Biotechnology (FIG. 0 L of BRM-Amp, 30° C., 18 hrs, uninduced, to verify expression performance.
Manufacturer:||BIOZOL|. Easy to identify: Includes green ~25 kDa and red ~75kDa reference bands. The b-chain eluted in the wash buffer. The combined fractions were reduced in vacuo by rotary evaporation at reduced pressure. The cells are re-suspended in the lysis reagent by vortexing.
1) to remove the 50 kDa insert. The 60 kDa BenchMark™ molecular weight marker protein includes six fused copies of a truncated E. coli thioredoxin protein (see U. In some embodiments, the invention provides pre-labeled molecular weight standard sets in which three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more of the labeled proteins of the set differ in size from one another by molecular weight increments that are multiples of 5 kDa, 10 kDa, 20 kDa, or 50 kDa. It was converted to the vinyl sulfone in order to react with the sulfhydryls of proteins for generating dyed marker proteins. 913 at 1 mg/ml concentration (according to the Swiss-Prot Protein Parameters tool). Lysine codons can be mutated to any nonlysine codons. The amount of protein and water added to the reactions was adjusted depending on the starting protein concentration. For example, a protein not related to a known naturally-occurring protein can be designed to be depleted in, preferably deficient in, a non-target amino acid and synthesized recombinantly or by chemical peptide synthesis. The pre-labeled protein standard set can include two or more, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more proteins that are selectively labeled on cysteine and are depleted in lysine, in which the selectively labeled proteins comprise one or more copies of an amino acid sequence depleted in lysine. In one embodiment of a kit, a pre-labeled standard set provided in a kit comprises a plurality of labeled proteins, in which one or more of the labeled proteins is selectively labeled on a first amino acid and lacks a second amino acid that is capable of reacting with a dye used to label the protein. 5 ml pre-stained ELITE Protein Ladder (10 x 0. The dried dye vinyl sulfone precursor was dissolved in 50 mL of water and transferred to a 100-200 mL round bottom flask equipped with a stir bar.
The invention provides individual pre-labeled proteins that migrate within 10%, within 7%, within 5%, within 4%, within 2. Ready-to-use: Supplied in a loading buffer for direct loading on gels. Migration of selectively labeled and unlabeled forms of a protein are preferably compared under electrophoresis conditions in which a the loading dye front migrates at least 6 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. Cell Mol Life Sci 77:2235-2253 (2020). Protein is eluted with Elution buffer (8M urea, 200 mM Imidazole, 0. Band Widths of Sharp Pre-stained Standard Proteins. 10) was cloned into the AvrII site. The calculated molecular weights of the proteins can be performed by curve-fitting of molecular weight to migration distances or point-to-point calculation. Sequences depleted in a non-target amino acid can be further selected based on the frequency of the target amino acid, e. g., cysteine. 100 μl of 10 mg/ml lysozyme (Calbiochem, San Diego, Calif., USA) solution in water was brought up to a volume of 1 ml with a final concentration of 50 mM Tris pH=8 and 0. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. The second amino acid is preferably a nontarget amino acid that can react with the labeling compound. The map of pTrc BH 50 kd and the sequence of the 50 kDa ORF encoded by the insert (SEQ ID NO:17) is shown in FIG.
Molecular weight marker. A dye used to label a selectively labeled protein of a pre-labeled protein standard set can be or comprise a chromophore, a fluorophore, or can be or comprise both a fluorophore and chromophore. The invention also includes nucleic acid constructs that encode proteins that comprise two or more copies of an amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein, in which all of the lysine codons have been deleted or changed to non-lysine codons. The collected fractions are analyzed by electrohoresis. To our knowledge, customised protocols are not required for this product. Titrate the pH to 7. In another example, glutamate can be a target amino acid, and aspartate can be a non-target amino acid. 13 depicts the reaction scheme for generating the vinyl sulfone form of Orange 16.
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