Preferably, a labeling compound used to label a protein standard has a high specificity for the reactive group of the target amino acid. Prestained Protein Ladder ab116028 is a three-color protein standard with 12 pre-stained proteins covering a wide range of molecular weights from 10 to 245 kDa. 2_B3 gel purified insert. 9), a truncated LacZ gene encoding a 100 kDa polypeptide (SEQ ID NO:40; FIG. The term "fluorophore" as used herein refers to a composition that is inherently fluorescent or demonstrates a change in fluorescence upon binding to a biological compound or metal ion, i. Novex sharp prestained protein standard chartered. e., fluorogenic.
The widths of the bands produced by the electrophoreses protein standard (peaks 2-13, corresponding to pre-stained protein bands on the gel), are provided in Table 7. Blue Protein Standard, Broad Range, New England Biolabs. 12 depicts a scheme for synthesizing 8-anilino-1-naphthalenesulfonic acid-aminophenyl vinyl sulfone (8-ANS-APVS). In the context of the present invention, "selectively labeled" means labeled predominantly on particular sites of a biomolecule. 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. 115: 1379-1387 (2005)) can be fused in any combination to provide protein standards.
Add 40 ml 1M sodium phosphate pH=7. Textile dyes are available from many commercial suppliers (for example, Burlington Chemical Co., Burlington, NC; Harneet Exports, Mumbai, India; Jagson Colorchem Ltd., Ahmadabed, India; Jaychem, Sanand, India; Omega Dyes, Goucestershire, UK; Dystar Textilfarben, Frankfurt, Germany; Kemtex, Chorley, UK). Approved for shipment on Wet or Dry Ice|. Provisional Application 60/820, 101 filed Jul. Designed for monitoring protein separation during PAGE and providing clear electro-transfer to commonly used membranes. Novex sharp prestained protein standard.html. The kit can also include instructions for use, or instructions for accessing protocols for use of the kit or its components via the internet. Ab116028 has been referenced in 16 publications. The method can be performed using curve-fitting or point-to-point calibration based on the migration of the at least two labeled standards or by calibration of protein standard migration normalized to dye front migration. Journal of Biological Chemistry 269: 15683 (1994)) or a sequence of one or more Bacillus megaterium spore proteins that lack cysteine residues (Setlow, Journal of Biological Chemistry 250: 8168 (1975)). All or a portion of a thioredoxin sequence can be used in making one or more pre-labeled protein standards. BRIEF DESCRIPTION OF THE DRAWINGS.
In this case, the expressed protein had a molecular weight that was closer to 160 kDa than to the expected 150 kDa. In preferred embodiments, all of the protein standards of the pre-labeled standard set are separated from one another such that the bands do not overlap and such that the widths of the bands on a gel of each of the electrophoresed proteins of the set having a molecular weight of 10 kDa or greater do not vary by more than 2-fold. The H2O wash is repeated, and then 300 μl of 50 mM Tris, 1% SDS pH=8 is added to the pellet. For example, the ratio of the number of residues of a target amino acid to molecular weight may be 4 residues per 10 kDa, or 0. Fisher Scientific is always working to improve our content for you. The invention includes protein standard sets that comprise one or more proteins selectively labeled on cysteine and depleted in lysine. 85 to obtain the width in millimeters. Pre-Labeled Proteins Having Consistent Ratios of a First Amino Acid to Molecular Weight. In some preferred embodiments, an amino acid sequence derived from a thioredoxin sequence differs from the naturally-occurring thioredoxin sequence by lacking lysine residues. 1B depicts the translated amino acid sequence of truncated E. coli bacterial thioredoxin having a C-terminal his tag on line 2 (SEQ ID NO:11) aligned with the same sequence in which all of the lysines have been changed to arginines and two cysteines have been added on line 1 (SEQ ID NO:12). Sequences depleted in a non-target amino acid can be further selected based on the frequency of the target amino acid, e. Novex sharp prestained protein standard edition. g., cysteine. For example, a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature, and that has not been intentionally modified in the laboratory is naturally-occurring. 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.
For Research Use Only. One tablet of inhibitor is used for every 50 ml solution. The sample can be in an aqueous solution, a viable cell culture or immobilized on a solid or semi solid surface such as a polyacrylamide gel, membrane blot or on a microarray. 4-aminophenyl-2-sulfonatoethyl sulfone (2. This clone was subsequently designated pTrc 260 kDa (FIG. Another factor contributing to poor resolution of pre-labeled proteins on electrophoresis gels is protein-to-protein variability in the ratio of the number of attached dye molecules to molecular weight. The sample is loaded on the column (about 20 ml of sample can be applied to 100 ml column bed volume). Highly Resolving Electrophoretic Separation of Pre-Labeled Protein Standards. The following procedures were used for the production of recombinant proteins for use as molecular weight standards. Reactive dyes and their preparation are well known in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra, (2002)). The LacZ gene was generated with Platinum® PCR Supermix High Fidelity PCR mix (Invitrogen; Carlsbad, Calif. ) using primers capped with Avr II restriction sites. For example, an engineered protein to be used for making pre-labeled protein standards can have one or more copies of an amino acid sequence with at least 70% or at least 80% identity with at least 20, at least 30, at least 40, or at least 50 contiguous amino acids of a thioredoxin sequence, in which lysine has been removed from the sequence by deletion or mutation of lysine codons in the nucleic acid sequence encoding the protein. As used herein, the articles "a, " "an" and "one" mean "at least one" or "one or more" of the object to which they refer, unless otherwise specified or made clear by the context in which they appear herein. 25 lpm air, 500 rpm agitation, and the pH is controlled to 6.
For example, using recombinant methods, sequences of proteins having at least a portion of the protein having fewer than one lysine per 10 kDa of protein, such as, for example, sequences encoding seed storage proteins of cereal crops (such as, for example, the zein proteins of maize, the gliadins of wheat), the L domain of HIV or Ebola viruses, or the WNK-1 and WNK-4 proteins (Coleman et al. 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 width of bands visible to the naked eye from proteins having a molecular weight of at least 20 kDa to less than 100 kDa range in width from 0. 5 kDa to greater than 250 kDa. The sequence of the insert was not directly determined. In some embodiments, a non-target amino acid has a different reactive group from the target amino acid.
The Blue Protein Standard, Broad Range is designed for observing protein separation during SDS-PAGE, verification of western transfer efficiency on membranes and for approximating the size of proteins. 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. A 100 mL round bottom flask was equipped with the appropriate sized egg-shaped stir bar. All gels were 8×8 cm "mini" gels from Invitrogen, Carlsbad, Calif., and electrophoresis conditions were those provided by the manufacturer. The components of the kit can in one or more containers, and two or more of the components of the kit can be provided in a common package (such as, for example, a box, rack, or jar). The appropriate amount of compound for any protein or other component is conveniently predetermined by experimentation in which variable amounts of the compound are added to the protein, the conjugate is purified (for example, using chromatography) to separate unconjugated compound and the protein-labeling compound conjugate is tested in its desired application. The pTrc 160 kDa construct was linearized with AvrII and gel purified. Alkylation was performed at 0. 8 using KOH or 5 M H3PO4.
Capping of Labeled Proteins. Lysine codons can be mutated to any nonlysine codons. Comprehensive - a wide range of molecular weight bands consisting of 12 proteins in the range of 3. In some aspects, a pre-labeled protein standard set can include one or more proteins not made by recombinant methods. The diazonium salt was transferred to an addition funnel and the diazonium salt solution was added to the solution of 8-ANS dropwise with stirring. Any of the amino acids cysteine, lysine, histidine, tryptophan, aspartate, glutamate, methionine, tyrosine, or asparagine can also be a non-target amino acid whose interaction with a labeling compound is sought to be reduced or eliminated when a protein is labeled on a first amino acid. Numerous fluorophores are known to those skilled in the art and include, but are not limited to coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene and xanthenes including fluoroscein, rhodamine and rhodol as well as other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM, Sep. 2002). 5 mg/ml solution of the 260 kDa standard protein.
All 7 lysine (K) amino acids were changed to arginine (R) at positions 4, 19, 52, 70, 83 and methionine (M) at position 36 to favor the binding of the dye molecules to cysteine rather than lysine. In a further aspect, methods are provided for characterizing one or more sample proteins using a pre-labeled protein standard set provided herein. 5 to 2 hours, or until the OD reaches 0. The sample is left to cool down to room temperature. 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. Invitrogen™ Novex™ Sharp Pre-stained Protein Standard by Thermo Fisher Scientific. The flask was charged with Reactive Orange 16 which was dissolved by the required volume of water. The volume of the column was at least 15 times the volume of the sample for the proteins labeled with Uniblue A, Orange 16 and Bodipy 530/550 dyes. 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.