Basic Protocol 1: Spectrophotometric Quantitation of Protein Carbonyls Using 2,4‐Dinitrophenylhydrazine Materials - DNPH solution (see recipe )
- Protein solution
- 2 M HCl
- 20% (v/v) trichloroacetic acid solution, ice‐cold (TCA; see recipe )
- 1:1 (v/v) ethanol/ethyl acetate
- 0.2% (w/v) SDS/20 mM Tris⋅Cl, pH 6.8 ( appendix 2A )
- Bicinchoninic acid protein assay kit (BCA; Pierce Co.)
- Bovine serum albumin (BSA)
- Benchtop centrifuge
- Branson 2200 sonicator
Support Protocol 1: Immunoblot Detection of Protein Carbonyls Materials - DNPH‐treated proteins ( protocol 1 )
- 5% (w/v) nonfat dry milk in Tris‐buffed saline with and without Tween‐20 (TBST; see recipe )
- Primary antibody (anti‐DNP antibody; Sigma)
- Secondary antibody: may be horseradish peroxidase–conjugated; select on the basis of nature of primary antibody
- Tris‐buffed saline with Tween‐20 (TBS and TBST; see recipe )
- ECL detection solution (Amersham)
- Minigel electrophoresis unit and transfer unit (Bio‐Rad; also see recipe for minigel recipes in unit 6.1 )
- Immobilon‐P membranes (Millipore)
- UV‐transparent plastic wrap
- X‐ray film
- Additional reagents and equipment for SDS‐PAGE (unit 6.1 ), staining gels with Coomassie blue (unit 6.6 ), and electroblotting proteins onto membranes (unit 6.2 )
Basic Protocol 2: Quantitation of Protein Carbonyls Derivatized with Tritiated Sodium Borohydride Materials - Protein solution
- 3 M Tris⋅Cl, pH 8.6 ( appendix 2A )
- 0.5 M EDTA, pH 8.0 ( appendix 2A )
- [3 H]NaBH 4 working solution (see recipe )
- 2 M HCl
- 20% (v/v) trichloroacetic acid solution, ice‐cold (TCA; see recipe )
- 1:1 (v/v) ethanol/ethyl acetate
- 0.2% (w/v) SDS/20 mM Tris⋅Cl , pH 6.8 ( appendix 2A )
- 0.5% (w/v) SDS/0.1 M NaOH
- BCA protein assay kit (Pierce)
- Scintisafe Plus 50% cocktail (Fisher Scientific.)
- Benchtop centrifuge
- Scintillation vials
CAUTION: Perform all incubations in a hood as tritium gas may be released during the reaction. When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D ). Support Protocol 2: Gel Electrophoretic Quantitation of Protein Carbonyls Derivatized with Tritiated Sodium Borohydride - Tritiated protein sample (see protocol 3 )
- 30% (v/v) hydrogen peroxide
- Additional reagents and equipment for SDS‐PAGE (unit 6.1 ) and staining gels with Coomassie blue R‐250 (unit 6.6 ).
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D ). Basic Protocol 3: Gel Electrophoretic Analysis of Protein Thiol Groups Labeled with [14C] Iodoacetamide Materials - Protein sample
- 1% (w/v) SDS/0.6 mM Tris⋅Cl buffer, pH 8.6 ( appendix 2A )
- 2‐mercaptoethanol, neat
- Nitrogen gas
- 500 mM [14 C] iodoacetamide, 1 µCi/ml (Amersham)
- 500 mM nonradiolabeled iodoacetamide
- SDS‐PAGE gels for Bio‐Rad Mini gel system (see recipe ):
- 10% resolving gel
- 4% stacking gel
- 10% (v/v) trichloroacetic acid (see recipe )
- Whatman 3MM filter paper
- X‐ray film
- Additional reagents and equipment for SDS‐PAGE (unit 6.1 ) and staining gels with Coomassie blue R‐250 (unit 6.1 ).
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D ). Basic Protocol 4: Quantification of Protein Dityrosine Residues by Mass Spectrometry Materials - Tissue sample
- o,o′ ‐dityrosine internal standards, labeled and unlabeled (see protocol 7 )
- Nitrogen gas
- 6 M HCl/1% (v/v) benzoic acid/1% (v/v) phenol
- Argon
- 10% and 0.1% (v/v) TCA solution (see recipe )
- 50 mM NaHPO 4 /100 µM diethylenetriamine pentaacetic acid (DTPA), pH 7.4
- 25% methanol
- 1:3 (v/v) HCl/n ‐propyl alcohol
- 1:4 (v/v) pentafluoropropionic anhydride/ethyl acetate
- Ethyl acetate
- n ‐propanol
- 0.1% (w/v) trifluoroacetic acid (TFA)
- Supelclean SPE reversed‐phase C‐18 column (Supelco)
- Hewlett Packard 5890 gas chromatography equipped with a 12‐m DB‐1 capillary column interfaced with Hewlett‐Packard 5988A mass spectrometer
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D ). Support Protocol 3: Preparation of o,o′‐Dityrosine standard Materials - Horseradish peroxidase (grade I; Boehringer Mannheim)
- 0.1 M borate buffer, pH 9.1 (see recipe )
- 5 mM L‐tyrosine (Sigma) or [13 C 6 ] L‐tyrosine (Cambridge Isotope Laboratories) in 0.1 M borate buffer, pH 9.1
- 30% (v/v) H 2 O 2
- 2‐mercaptoethanol
- 0.01 M NaOH ( appendix 2A )
- 200 µM borate buffer, pH 8.8: diluted from 0.2 M borate buffer (see recipe ) with H 2 O
- 2.75 × 19.5–cm DEAE cellulose chromatography column (Bio‐Rad)
- 20 µM NaHCO 3 , pH 8.8 (see recipe )
- Concentrated and 100 mM formic acid
- 100 mM NH 4 HCO 3
- Benchtop centrifuge
- 4 × 34.5–cm BioGel P‐2 column (200‐4‐mesh; Bio‐Rad)
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D ). Support Protocol 4: Analysis of Protein‐Bound Notrotyrosine by a Competitive ELISA Method Materials - 10 µg/ml nitro‐bovine serum albumin (nitro‐BSA; Alexis Biochemicals) in plate coating buffer
- Nitro‐BSA standard (see recipe )
- ELISA buffers (see recipe ):
- Plate coating buffer
- 1× phosphate‐buffered saline/Tween 20 (PBST)
- Blocking buffer
- 1× diethanolamine (DEA) buffer
- Protein sample
- Primary antibody: mouse anti‐nitrotyrosine antibodies (Upstate Biotechnology)
- Secondary antibody: rabbit anti‐mouse IgG conjugated with alkaline phosphatase
- Tris‐buffered saline/Tween‐20 (TBST; see recipe )
- 1 mg/ml p ‐nitrophenyl phosphate (5‐mg tablets; Sigma) in DEA buffer (see recipe for ELISA buffer)
- 96‐well ELISA plates
- Plastic wrap
- Plate reader
Basic Protocol 5: Enzymatic Analysis of Isoaspartate Formation Materials - 0.2 M Bis‐Tris buffer, pH 6.0 (see recipe )
- 10 µM [3 H]methyl‐S‐adenosyl‐L‐methionine (5 to 15 Ci/mmol; [3 H] SAM; NEN)
- Protein‐L‐isoaspartyl methyltransferase (PIMT; Promega Corporation or purified from a known source)
- 0.2 M NaOH ( appendix 2A )
- Safety‐Solve II counting fluor (Research Products International)
- Sponge plugs (Jaece Industries): cut into small pieces
- Scintillation vials with extra caps
CAUTION: [3 H]methanol is volatile at room temperature. Perform all reactions under a hood. When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D ). |
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