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Evaluation of the Cytochrome b5/Cytochrome b5 Reductase Pathway
Publication Name:
Current Protocols in Toxicology
Unit Number:
UNIT 4.16
DOI:
10.1002/0471140856.tx0416s24
Online Posting Date:
June, 2005 Abstract
NADH cytochrome b
Keywords: Cytochrome b
Table of Contents
- Unit Introduction
- Basic Protocol 1: Ferrihemoglobin Reduction as a Marker of Cytochrome b
5 Reductase Activity in Erythrocytes - Support Protocol 1: Preparation of RBC Lysate
- Alternate Protocol: Ferrihemoglobin Reduction Activity in Mononuclear Leukocytes
- Support Protocol 2: Isolation of Peripheral Blood Mononuclear Cells
- Basic Protocol 2: Quantitative Reverse Transcriptase Polymerase Chain Reaction for Expression of Cytochrome b
5 Reductase and Cytochrome b5 - Basic Protocol 3: Expression of Human Recombinant Soluble Cytochrome b
5 Reductase or Cytochrome b5 in E. Coli - Reagents and Solutions
- Commentary
- Literature Cited
- Tables
Materials
Basic Protocol 1: Ferrihemoglobin Reduction as a Marker of Cytochrome b5 Reductase Activity in Erythrocytes
Materials
- 0.027 M disodium EDTA, pH 7.0: dissolve 392 mg Na
2 EDTA in 50 ml H2 O; adjust pH to 7.0 using 1 M NaOH - 50 mM sodium citrate buffer, pH 4.7 (see recipe)
- 0.5 mM potassium ferricyanide: dissolve 3.292 mg K
3 Fe(CN)6 in 20 ml H2 O (prepare fresh; protect from light) - 1.224% (w/v) human hemoglobin (see recipe)
- RBC lysate from subject of interest, diluted 1:20 (see Support Protocol 1)
- 2 mM NADH: dissolve 2.836 mg NADH in 2 ml H
2 O (degrades in solution at room temperature; prepare fresh within 1 hr of use.)
- 1.5-ml amber microcentrifuge tubes
- Quartz spectrophotometer cuvettes
- Spectrophotometer
Support Protocol 1: Preparation of RBC Lysate
Materials
- Heparinized blood
- Phosphate-buffered saline, pH 7.4 (PBS; appendix 2A or purchase from Sigma)
- Stabilizing solution (see recipe)
- Tabletop centrifuge
- 1.5-ml amber microcentrifuge tubes
- Co-oximeter (Instrumentation Laboratory; http://www.ilus.com/)
Alternate Protocol: Ferrihemoglobin Reduction Activity in Mononuclear Leukocytes
Additional Materials (see Basic Protocol 1)
- Leukocytes (PBMC; see Support Protocol 2)
- Bath sonicator
- Bradford protein assay kit (Bio-Rad)
Support Protocol 2: Isolation of Peripheral Blood Mononuclear Cells
Materials
- Heparinized whole blood from subject of interest, freshly obtained
- Hanks' Balanced Salt Solution (HBSS; appendix 2A) containing 5 mM EDTA, pH 7.4
- Lymphocyte Separation Medium (LSM; Mediatech Inc.)
- ACK lysis buffer (Cambrex Biosciences; http://www.cambrex.com)
- Phosphate-buffered saline (PBS; appendix 2A)
- 50-ml conical centrifuge tubes
- Tabletop centrifuge
Basic Protocol 2: Quantitative Reverse Transcriptase Polymerase Chain Reaction for Expression of Cytochrome b5 Reductase and Cytochrome b5
Materials
- Source for RNA: e.g., peripheral blood mononuclear cells (PBMC; see Support Protocol 2)
- RNA-preserving solution: e.g., RNAlater (optional; Ambion)
- Phosphate-buffered saline, pH 7.4 (PBS; appendix 2A)
- RNA isolation kit: e.g., RNAqueous-4PCR (Ambion)
- DNase/RNase-free H
2 O - cDNA preparation kit: e.g., SYBR Green PCR/RT-PCR kit (Applied Biosystems)
- Primers (prepare in DNase/RNase-free H
2 O; store in aliquots at –20°C):- 6.66 µM 18S rRNA forward primer: 5¢-CGCCGCTAGAGGTGAAATTCT-3¢
- 6.66 µM 18S rRNA reverse primer: 5¢-CGAACCTCCGACTTTCGTTCT-3¢
- 10.0 µM b
5 R forward primer: 5¢-AGGGCAAAGGGAAGTTCGCCAT-3¢ - 10.0 µM b
5 R reverse primer: 5¢-ACAGACTTCACTGTCCTGATGATA-3¢ - 10.0 µM cyt b
5 forward primer: 5¢-CTGCACCACAAGGTGTACGA-3¢ - 10.0 µM cyt b
5 reverse primer: 5¢-ACCTCCAGCTTGTTCCCTTA-3¢
- UV spectrophotometer
- DNase/RNase-free pipet tips with filters
- 250-µl clear DNase/RNase-free tubes
- Standard thermal cycler (for reverse transcription)
- 96-well PCR plates (e.g., Bio-Rad)
- Real time PCR cycler (e.g., Bio-Rad iCycler)
- Optical quality sealing tape (e.g., Bio-Rad)
- Tabletop centrifuge with microtiter plate carrier
Basic Protocol 3: Expression of Human Recombinant Soluble Cytochrome b5 Reductase or Cytochrome b5 in E. Coli
Materials
- pCR T7 TOPO TA expression vector (Invitrogen): NT or CT
- Chemically competent E. Coli: e.g., One Shot Chemically Competent Cells [BL21(DE3) cells] from Invitrogen
- SOC medium (supplied with the One Shot Chemically Competent Cells from Invitrogen)
- LB broth (see recipe)
- 100 mg/ml ampicillin stock solution (see recipe)
- 10 mM IPTG (see recipe)
- Binding buffer, pH 8.0 (see recipe)
- Complete Mini Protease Inhibitor Cocktail tablets (Roche Diagnostics; optional)
- Lysozyme (Sigma; optional)
- Ni-NTA agarose resin (25 ml ethanol suspension; Invitrogen)
- Elution solutions 1, 2, and 3 (see recipe)
- SDS-PAGE loading buffer: add 50 µl 2-mercaptoethanol (Sigma) to 950 µl Laemmli sample buffer (Bio-Rad); prepare fresh for each purification
- Tris-HCl precast polyacrylamide gels (Bio-Rad) compatible with Mini PROTEAN 3 Electrophoresis Cell: 12% gel (for b
5 R expression) or 15% (for cyt b5 expression) - 1× Tris-glycine electrode buffer (see unit 2.2 for 10× buffer)
- Phosphate-buffered saline, pH 7.4 (PBS; appendix 2A or purchase from Sigma)
- Bradford protein assay kit (Bio-Rad)
- 2 mM hemin (see recipe)
- QIAprep Plasmid Prep kit (Qiagen)
- 42°C water bath
- 37°C warm room with rotary shakers
- 100- and 2000-ml Erlenmeyer flasks, sterile
- 250-ml culture tubes
- Refrigerated centrifuge capable of holding 250-ml culture tubes, capable of 12,000 × g
- Safety goggles and ear plugs
- Sonic Dismembranator with microtip attachment (Fisher)
- 15- or 50-ml conical polypropylene centrifuge tubes
- 10-ml Poly-Prep columns (Bio-Rad)
- 0.5-ml microcentrifuge tubes
- Mini PROTEAN 3 Electrophoresis Cell (Bio-Rad)
- 95°C heating block
- Dialysis cassettes (Pierce): 10,000 MWCO (for b
5 R expression) or 3,500 MWCO (for cyt b5 expression) - 1000-ml beaker
- 15-ml Centriplus centrifugal concentrators (Millipore): 10,000 NMWL (for b
5 R expression) or 3,000 NMWL (for cyt b5 expression)
- Additional reagents and equipment for protein electrophoresis and gel staining (appendix 3F) and dialysis and concentration of protein solutions (appendix 3H)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.
Looking for materials? Suppliers Guide
Literature Cited
| Literature Cited | |
| Beutler, E. 1975. A Manual of Biochemical Methods. 2nd ed. Grune & Stratton, New York. | |
| Giordano, S. and Steggles, A. 1991. The human liver and reticulocyte cytochrome b | |
| Hildebrandt, A. and Estabrook, R. 1971. Evidence for the participation of cytochrome b | |
| Hultquist, D. and Passon, P. 1971. Catalysis of methaemoglobin reduction by erythrocyte cytochrome b | |
| Ito, A., Hayashi, S., and Yoshida, T. 1981. Participation of a cytochrome b | |
| Kurian, J., Bajad, S., Miller, J., Chin, N., and Trepanier, L. 2004. NADH cytochrome b | |
| Leroux, A., Vieira, L., and Kahn, A. 2001. Transcriptional and translational mechanisms of cytochrome b | |
| Oshino, N., Imai, Y., and Sato, R. 1971. A function of cytochrome b | |
| Shirabe, K., Landi, M., Takeshita, M., Uziel, G., Fedrizzi, E., and Borgese, N. 1995. A novel point mutation in a 3¢ splice site of the NADH-cytochrome b | |
| Tomatsu, S., Kobayashi, Y., Fukumaki, Y., Yubisui, T., Orii, T., and Sakaki, Y. 1989. The organization and the complete nucleotide sequence of the human NADH-cytochrome b | |
| Internet Resources | |
| http://www.ambion.com/techlib/basics/rtpcr | |
| Web site with basic background for the qRT-PCR technique. | |
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