Literature Cited Jfj
Ackers, G.K. 1970. Analytical gel chromatography of proteins. Adv. Protein Chem. 24 342-443. Allet, B., Payton, M., Mattaliano, R.J., Gronenborn, A.M., Clore, G.M., and Wingfield. P.T. 1988. Purification and characterization of the DNA-binding protein ner of bacteriophage mu. Gene 65 259-268. Ballery, N., Desmadril, M., Minard, P., and Yon, J.M. 1993. Characterization of an intermediate in the folding pathway of phosphoglycerate kinase Chemical reactivity of genetically introduced cysteinyl...
Dynamic Light Scattering Analysis Of Protein Solutions
Whereas static light scattering is concerned with the time-averaged scattering intensity properties, dynamic light scattering is concerned with time fluctuations in intensity caused by motions of macromolecules and macromolecular assemblies. Whereas lasers are highly desirable for static light scattering because of their high intensity, collimation, and monochromaticity with dynamic light scattering they are mandatory because of the requirement for spatial and time temporal coherence light has...
Titration Of Vaccinia Virus Stocks By Plaque Assay
Serial dilutions of the trypsinized virus stock see Basic Protocol 3 are used to infect the appropriate cell line. After several days growth, the medium is removed and the cells are stained with crystal violet. Plaques appear as 1- to 2-mm-diameter areas of diminished staining due to the retraction, rounding, and detachment of infected cells. Additional Materials also see Basic Protocols 1 and 3 BS-C-1 cells from confluent monolayer culture see Basic Protocol 1 Virus stock see Basic Protocol 3...
Background Information Xbi
Prenylation of proteins is a widespread post-translational modification that occurs at C-ter-minal motifs of the type CXXX, CXC, or CC where C is cysteine and X is any amino acid . Recent reviews give a thorough description of this field Clarke, 1992 Giannakouros et al., 1993 . Either the 15-carbon isoprenoid farnesol or the 20-carbon isoprenoid geranylgeraniol see Fig. 14.3.1 can be added, depending on the exact sequence at the C-terminus. CX1X2X3 motifs where X3 is a small residue e.g.,...
Prenylation Of Proteins During In Vitro Translation
Commercially available reticulocyte lysates contain protein prenyl transferases, so proteins can be labeled during in vitro translation of mRNA using 3H mevalonic acid Hancock et al., 1991 Newman et al., 1992 Giannakouros et al., 1993 , which is converted to prenyl diphosphates in situ. When 3H mevalonic acid is used, the identity of the incorporated label as farnesyl or geranylgeranyl must be experimentally determined by cleavage of the label followed by chain-length analysis using the method...
Info Eue 1
recipes see Reagents and Solutions . Lipid substrates should never be dried to completion in plastic tubes because they are often irreversibly adsorbed to the surface. The ability to detect prenylation and or car-boxyl-methylation of any given protein using these methods will depend on a number of factors the ability of the cells to take up radiolabeled compounds and incorporate them into metabolic precursors the sensitivity of the cells to hydroxymethylglutaryl coenzyme A HMG-CoA reductase...
Info Nth
keep the protein soluble, because many proteins are least soluble close to their isoelectric points. In addition, pH can be used as a means to manipulate the stability of the folded protein. Many proteins are too stable to be unfolded in 8 M urea at neutral pH, but can be unfolded by urea under alkaline or acidic conditions. Several different buffers have been used successfully for urea-gradient gels see Reagents and Solutions . Ideally, the ionic strength of the buffer solution should be as...
Info Buj
aAll resins are from Pharmacia Biotech except Bio-Gel A-5m, which is from Bio-Rad. The Sepharose and Bio-Gel matrices are normally run under low pressure all other resins can be run under low or medium pressure. Medium pressure is achieved using one of the chromatography pumps indicated in Basic Protocol 2 the pumps are normally included in the Pharmacia Biotech FPLC or BioPilot systems. bData on the fractionation range in the unfolded state refer to proteins unfolded with guanidine-HCl...
Info Uxw
of salt ions are removed by the mixed bed 1 the supporting electrolyte i.e., the free salt in the solution and 2 salt ions that are counterions, which are in effect bound to the protein if the protein is not isoionic. If the pH of the starting solution is below the isoionic point, the protein is a cation with anion counterions, commonly Cl-. If the pH is greater than the isoionic point, the protein is a net macroanion with cationic counterions, commonly K or Na . In the mixed bed, cationic and...
Preparation Of Sheared Salmon Sperm Carrier Dna
This protocol generates high-quality sheared salmon sperm DNA sssDNA for use as carrier in transformation Basic Protocol 2 . This DNA is also suitable for other applications where high-quality carrier DNA is needed e.g., hybridization . This protocol is based on Schiestl and Gietz 1989 . For more details of phenol extraction or other DNA purification methods, consult appendix 4e. High-quality salmon sperm DNA e.g., sodium salt from salmon testes, Sigma or Boehringer Mannheim , desiccated TE...
Intramolecular Disulfide Formation By Potassium Ferricyanide Oxidation
Efficient oxidation rates have been reported, especially in the oxytocin and somatostatin families, when using potassium ferricyanide, a relatively mild inorganic oxidizing reagent. Because K3Fe CN 6 is slightly light sensitive, reactions are best conducted in the dark. The indicated order of addition, i.e., peptide to oxidizing agent, is designed to favor intramolecular cyclization because the concentration of free thiol species is kept minimal. Oxidation side products are possible when Met or...
DETECTION OF PROTEINS MODIFIED BY OGlcNAc USING ANTIBODIES
Recently Comer et al. 2001 showed that an antibody CTD 110.6 raised against the glycosylated C-terminal domain of the RNA polymerase II large subunit was a general O-GlcNAc antibody. Unlike many lectins, CTD 110.6 shows little cross-reactivity toward terminal GlcNAc on complex glycans. Several other antibodies, HGAC 85 Turner et al., 1990 and RL2 Snow et al., 1987 , have been reported as O-GlcNAc specific antibodies. However, these antibodies only recognize a subset of O-GlcNAc modified...
Phenylisothiocyanate Derivatization
PTC-AAA typically identifies the sixteen common amino acids in peptide protein hydro-lysates, including proline. The derivatization reaction is relatively tolerant to variations in pH and molar ratios of reagents to sample, but a slow, variable degradation of the derivatives can occur in solution at room temperature. The derivatization protocol may also be successfully performed with ethanol in place of methanol, and triethylamine TEA substituted for N,N-diisopropylethylamine DIEA , but...
Info Tqp
are analyzed, such as unseparated protein digests. For example, in tryptic digest mixtures, the C-terminal arginine-containing components are preferentially detected. Therefore, to obtain better detection of most of the components in such a mixture, spectra should be acquired using different matrix solution conditions and different matrices. Contaminants such as salts and detergents should be lt 50 mM. When crystallization is impaired due to the presence of contaminants, the matrix sample...
Info Aby
Figure 5.13.1 Homologous recombination between a transfected plasmid and the vaccinia virus genome. TKL and TKR are vaccinia virus DNA sequences flanking the foreign gene. p11 and pj5 are promoters. 1. Subclone the gene of interest into the multiple cloning site MCS in pSC11, pRB21, pSC65, pLW9, or other suitable vector and isolate plasmid appendix 4c . 2. Seed a 25-cm2 flask with 1 x 106 CV-1, BS-C-1, BHK-21, or CEF cells in complete MEM-10 medium. Incubate to near confluency usually overnight...
Preparative and Analytical Electrophoretic Separation see Basic Protocol 4 and
Separation of small organelles and macromolecular assemblies on the basis of distinct surface charge densities. 1. Although electrophoretic separations have not been used widely in organelle purification, the preparative technique Basic Protocol 4 shows substantial promise because it is reasonably easy, not expensive, and as reported by its originators L. Rome, pers. comm. is capable of handling relatively large samples i.e., 50 mg protein . Furthermore, preliminary findings suggest that...
Covalent Attachment Of Biotin To Lysines
This protocol is a specialized version of the succinimidyl ester amidation reaction described in Basic Protocol 1 of unit 15.2. The reagent used, NHS-LC-biotin see Fig. 3.6.1 , has an N-hydroxysuccinimide NHS ester group to increase reactivity with primary amines and a long-chain LC spacer arm to extend the biotin from the protein surface, thus improving binding of subsequent reagents e.g., avidin . The protein of interest is incubated with NHS-LC-biotin. Glycine is added to stop the reaction,...
Prepare the agarose gel
1. Wash the agarose gel Sepharose CL-4b, 10 g wet weight on a sintered glass funnel successively with 100 ml of each of the following ice-cold solutions 30 70 v v acetone water 70 30 v v acetone water 100 reagent-grade acetone anhydrous acetone anhydrous acetonitrile. 2. Suspend the agarose gel in 12 ml of 4 C anhydrous acetonitrile containing 800 mg p-nitrophenyl chloroformate. Slowly add 3 equivalents of dimethylaminopyridine in anhydrous acetonitrile and stir the mixture for 1 hr at 0 to 4...
Info Ifb
Figure 2.3.4 Secondary structure prediction for human profilin I using the methods of Chou and Fasman and GOR as implemented by GCG PeptideStructure algorithm and plotted using Plot-Structure utility. A Predicted secondary structure and B numerical output for residues 1 through Protein 35 Secondary Structure Prediction with the Chou and Fasman, GOR, PHD, and PSA methods Fig. 2.3.9 . Analysis of the Chou and Fasman profiles was enhanced by displaying smoothed hydrophobicity profiles and...
Background Information Yib
The transfer of proteins from polyacrylamide gels onto blot membranes offers many benefits. Transferred proteins can be eluted from the membrane, probed with antibodies immunoblotting , used for N-terminal protein sequencing as well as other structural analyses including amino acid analysis, protease cleavage unit 11.2 , and chemical cleavages unit 11.5 , or stained by a variety of highly sensitive techniques. In addition, several manipulations can be performed on the same blot by loading...
Literature Cited Uyh
Aguilar, M.I., Hodder, A.N., and Hearn, M.T.W. 1985. Studies on the optimization of the re-versed-phase gradient elution of polypeptides Evaluation of retention relationships with P-en-dorphin related polypeptides. J. Chromatogr. 327 115-138. Alpert, A.J. 1990. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr. 499 177-196. Anspach, B., Unger, K.K., Davies, J., and Hearn, M.T.W. 1988. Affinity chromatography with...
Info Yrd
Coomassie blue and silver staining are the two most common approaches used for protein detection in gels with fixation. Silver staining relies on binding of silver to amino acid side chains, primarily sulfhydryl and carboxyl groups, and Coomassie blue binds nonspecifi-cally to proteins. Silver staining is typically about 10 to 100 times more sensitive than Coomassie blue staining but it is more complex and time-consuming. There are many variations of silver stain methods both in the scientific...
Homogenization Of Tissue Culture Cells
Gentle homogenization conditions should be used to limit possible damage to endosomal elements, particularly when using fluid-phase markers. Clearly, the markers should remain entrapped in vesicles latent after homogenization. In addition, harsh conditions should always be avoided to limit the breakage of lysosomes and consequent proteolysis by released hydrolases. Cells grown for 14 to 16 hr are easily homogenized, so each step of the homogenization process should be monitored using a...
Info Rix
Two-dimensional gel electrophoresis, using isoelectricfocusing followed by SDS-PAGE, is the single most powerful analytical method currently available for separating complex protein mixtures such as whole-cell or tissue extracts. It is therefore a valuable method for following disease-related changes or for detecting changes in protein expression under diverse experimental conditions. To achieve maximum reproducibility between samples to be compared, multiple gels should be cast and run...
Info Pss
440 g each of two synthetic oligonucleotides with desired binding site see Support Protocol 1 or commercial HPLC-purified TE buffer, pH 7.8 appendix 2e 10x T4 polynucleotide kinase buffer see recipe 20 mM ATP Na salt , pH 7.0 150 mCi ml y-32P ATP 6000 Ci mmol 10 U l T4 polynucleotide kinase New England Biolabs 10 M ammonium acetate appendix 2e 25 24 1 v v v phenol chloroform isoamyl alcohol 24 1 v v chloroform isoamyl alcohol 3 M sodium acetate appendix 2e 100 and 75 ethanol 10x linker kinase...
Acidbased Coomassie Blue G250 Staining
The acid-based Coomassie brilliant blue G-250 staining procedure fixes and stains the gels simultaneously and, unlike the first basic and alternate protocols, does not require a destaining solution. Color intensifies when the gels are placed in water after the staining period, and as little as 50 to 100 ng protein per band can be detected. Acid-based Coomassie blue G-250 which is also used in a colloidal state rather than a solution, staining has a sensitivity similar to that of the basic...
Basic Protocol 2 Uij
Additional reagents and equipment for standard denaturing SDS-PAGE see Basic Protocol 1 1. Assemble each gel sandwich by stacking, in order, the notched Hoefer Pharmacia or small rectangular Bio-Rad plate, 0.75-mm spacers, and the larger rectangular plate. Be sure to align the spacers properly with the ends flush with the top and bottom edge of the two plates when positioning the sandwiches in the multiple gel caster Fig. 10.1.4 . The protocol described is basically for the Hoefer Pharmacia...
Preparing Endtoside Chain Cyclic Peptides Via A Thiazolidine Or Oxime Linkage
In these two examples Pallin and Tam, 1995 Botti et al., 1996 , the precursor contains a Lys residue which is introduced as an Fmoc-Lys Mtt for attaching Ser through its side chain. The sequences are peptide antigens ranging from 5 to 26 amino acids in length and are derived from the V3 loop of gp120, HIV-1. The 1,2-amino alcohol function of Ser acts as a masked aldehyde and can be converted to glyoxyaldehyde after treatment with NaIO4. A Cys StBu moiety is incorporated at the N-terminus to...
Cleavage At Asngly Peptide Bonds With Hydroxylamine
Hydroxylamine cleaves proteins on the C-terminal side of Asn residues in asparagine-glycine Asn-Gly peptide bonds. For proteins with multiple Asn-Gly peptide bonds, fragments generated from this reaction can be incomplete i.e., contain an internal Asn-Gly . After digestion with hydroxylamine, resulting peptide fragments are fractionated by HPLC or electrophoresis prior to further characterization. 1 to 50 g lyophilized protein sample in a 0.5-ml capped microcentrifuge tube 1.8 M hydroxylamine...
Unit 45
Selective precipitation of proteins Rothstein, 1994 can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. Except for antibody-mediated precipitation see unit 10.8 , selective precipitation methods are usually not protein-specific the process depends on the physical and or chemical...
Reagents And Solutions Jdo
Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2E for suppliers, see SUPPLIERS APPENDIX. Buffer pH and conductivities are for solutions at 4 to 6 C. Units of conductance are given in siemens S Q-1 . Anion-exchange buffer 50 mM Tris-Cl, pH 8.5 Dilute 1 M Tris-Cl, pH 8.0 20-fold with water and adjust to pH 8.5 with NaOH. Make immediately before use. Conductivity of the solution is 1.57 mS cm. Cation-exchange buffer, 10x 15 mM...
Info Klj
presence of enzymatically prepared product. A decent understanding of the possible presence of product inhibition in initial-rate experiments is always useful to the protein chemist. Enzymes often undergo a slow inactivation. Usually, the rate of this inactivation is accelerated by dilution of the stock enzyme preparation. Efforts have to be made to eliminate or lessen the loss of enzyme activity with time. Often, multisubstrate enzymes are stabilized by the presence of a substrate. For...
Purify fractions further
Example For parotid-gland secretion granule fraction 8. Load the adjusted granule fraction into centrifuge tubes and recentrifuge under the same conditions as used in the first run. This repeat centrifugation will float much of the residual contaminants away from the granule fraction. 9. Collect the granule fraction manually with a pipet. 10. If desired, process the purified granule fraction as a single organelle population by diluting with 3 vol homogenization medium, then pelleting the...
Info Xus
tPA leader peptide has been used for efficient translocation Burke et al., 1986 . The protein is folded and glycosylation is initiated in the ER. Detailed discussion of the intracellular factors involved in protein translocation are given in a number of excellent reviews Gething and Sambrook, 1992 Rothman and Orci, 1992 Wirth and Hauser, 1993 Rothman, 1994 . The protein begins to fold cotranslationally in the ER. The ER provides an environment optimized for protein folding and is distinguished...
ISOELECTROFOCUSING USING IMMOBILIZED pH GRADIENT GEL STRIPS
In immobilized pH gradient IPG gels, the ampholytes are covalently linked to the acrylamide matrix, which facilitates production of highly reproducible gradients as well as very narrow pH gradients for optimal resolution of minor charge differences. A variety of precast gels and all the necessary equipment are commercially available from Hoefer Pharmacia. Equipment and chemicals are also available for the user to cast gels in the laboratory see Support Protocol 3 , although precast gels are...
Info Jeh
The quantities directly measured by a light scattering photometer are voltages and not light scattering intensities. For this reason, the instrument has to be calibrated usually with a strong Rayleigh scatterer with a known Rayleigh ratio such as toluene Stacey, 1956 Johnson and McKenzie, 1977 . Measurement of the scattered and incident light intensities Ie and I0, respectively then facilitates calculation of the calibration constant, which is then used to calculate Rayleigh ratios for sample...
Desalting The Sample After Cysteine Modification
Except for the in situ method Basic Protocol 9 , all of the protocols presented leave the protein in the presence of high salt and reaction by-products which are usually not compatible with subsequent analytical techniques. This problem may be remedied by one of the following desalting methods. The methods are presented in increasing order of technological complexity. Protein recovery is a concern because small quantities of protein may be lost in dialysis or gel filtration. Modern...
Expression Using Glucoserepressible Adh2 Vectors
The ADH2 promoter is repressed 100-fold by glucose. To maintain repression, cells must always be grown in excess glucose e.g., 8 w v because normal concentrations e.g., 2 can become depleted. It is also preferable to store transformants on plates containing 8 glucose. Inductions can be carried out by first growing cells in medium containing excess glucose, then inducing by changing to fresh medium containing a nonfermentable carbon source such as raffinose. Additional Materials also see Basic...
Colorimetric Assay For Pyroglutamate Aminopeptidase Activity
Pyroglutamate aminopeptidase activity is not particularly stable. One cause of failure to remove an amino-terminal blocking group may be lack of activity in the enzyme stock solution. The following protocol describes the method used by Boehringer Mannheim and several authors for monitoring enzyme activity based on generation of P-naphthy-lamine from L-pyroglutamyl-P-naphthylamide. An alternative method for monitoring enzyme activity assaying for the hydrolysis of a synthetic peptide with...
Hydrolysis with 4 N Methanesulfonic Acid for Quantification of Tryptophan
This alternative procedure, hydrolysis with 4 N methanesulfonic acid, is adapted from Hsi and Yamane 1994 . Response factors for Trp are determined from 4 N methanesul-fonic acid hydrolysates of standard proteins and concurrent hydrolysis and analysis of control and unknown samples. Additional Materials also see Basic Protocol 1. Add 30 l of 4 N methanesulfonic acid to the dried sample in a clean 6 x 50-mm tube. Seal sample tubes in bulge-modified 40-ml screw-cap vials and alternately flush...
Continuous Sdspage
With continuous SDS-PAGE, the same buffer is used for both the gel and electrode solutions. Although continuous gels lack the resolution of the discontinuous systems, they are extremely versatile, less prone to mobility artifacts, and much easier to prepare. The stacking gel is omitted. Additional Materials also see Basic Protocol 1 Separating gel solution Table 10.1.7 2x and 1x phosphate SDS sample buffer see recipe 1x phosphate SDS electrophoresis buffer see recipe 1. Prepare and pour a...
Info Sdp
aThe device with the highest sensitivity and greatest dynamic range for a visualization method is marked with a , other devices that can detect this visualization method are indicated with a , and devices that are not suitable for a visualization method are indicated with a . A indicates that only some devices of this type can be used with this visualization method. aThe device with the highest sensitivity and greatest dynamic range for a visualization method is marked with a , other devices...
Nondenaturing lysis buffer
1 w v Triton X-100 store at room temperature in dark 1 mM PMSF store 100 mM stock in 100 ethanol up to 6 months at -20 C 2 g ml leupeptin store 10 mg ml stock in H2O up to 6 months at -20 C 1 mM AEBSF, added fresh from a 0.1 M stock solution in H2O, can be used in place of PMSF. AEBSF stock can be stored up to 1 year at -20 C.
Info Ssr
Smith, D.L., Deng, Y., and Zhang, Z. 1997. Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry. J. Mass Spectrom. 32 135-146. Speir, J.A., Munshi, S., Wang, G., Baker, T.S., and Johnson, J.E. 1995. Structures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscopy. Structure 3 63-78. Th venon-Emeric, G., Kozlowski, J., Zhang, Z., and Smith, D.L. 1992. Determination of amide...
Reduce disulfide bonds of digest with TCEP
10. Add an equal volume of reducing solution, pH 8.0, to the 1.5-nmol aliquot. Mix and blanket reaction mixture with argon. Although TCEP can function at acidic pH, reduction of disulfide bonds is most effective at pH 7 to 8. An inert atmosphere prevents reoxidation of cysteines to disulfides. 11. Incubate the mixture 1 hr at 37 C. 12. Adjust the pH of the reduced tryptic digest by adding TFA to a final concentration of 2 . This reduces the pH to 2 for RP-HPLC analysis.
Info Rpn
Constant-temperature water bath Two-head peristaltic pump 10- l glass capillary pipet optional Clay-Adams screw-top loader optional Whatman 3MM filter paper or equivalent Additional reagents and equipment for digesting DNA with restriction endonucleases, DNA labeling, agarose and nondenaturing polyacrylamide gel electrophoresis, recovery of DNA from gels, oligonucleotide synthesis, PCR, ethanol precipitation, ethidium bromide dot quantitation, and autoradiography see Ausubel et al., 1998, or...
Info Kak
as the microscopic and global hydrophobicity. These important factors ultimately determine the selection of the optimal separation conditions for the resolution of peptide and protein mixtures. Table 8.7.1 can be used to evaluate the impact of amino acid composition on retention behavior. For example, this information can be used to direct the choice of eluent composition or the gradient range in RP-HPLC to assess the impact on retention of amino acid substitution or deletion with small...
Abi Model 420a Manual
Figure 11.9.13 Identification of CRALBP from PTC-AAA. The raw PTC-AAA data in Figure 11.9.12 from analysis of -27 pmol recombinant human CRALBP was converted to mole and entered into the PROPSEARCH and ExPASy database query programs for identification of the protein. The following mole data were entered Arg 7.07, Met 2.05, Glx 17.32, Ile 3.31, Ser 4.62, Leu 10.31, His 1.72, Phe 7.44, Gly 7.46, Lys 5.01, Thr 4.84, Asx 7.37, Ala 8.22, Pro 4.32, Tyr 2.86, Val 6.09, sum 100.01. The top ten scores...
Rcsb Pdb
The Research Collaboratory for Structural Bioinformatics RCSB Protein Data Bank PDB is a comprehensive database of three-dimensional protein and nucleic acid structures determined by X-ray crystallography, NMR, cryoelectron microscopy, and theoretical modeling Berman et al., 2000 . It is maintained by Rutgers, The State University of New Jersey the San Diego Supercomputer Center SDSC at the University of California, San Diego and the National Institute of Standards and Technology NIST . The PDB...
Info Brd
well as the number of such substituents. In practice, one expects to initially experiment with 3 to 5 such ligands to find a ligand suitable for investigation in more detail. Because matrix ligands generally are quite protective of proteins with which they coprecipitate, it may not be necessary to always develop the coprecipitation stage of protein isolation at cold temperatures. Indeed, the method often interfaces well with a heat-shock step to clear out unwanted proteins susceptible to heat...