Methods

The following topics are described briefly:

I. Tissue preparation;
II. Fluid sampling;
III. 1-and 2D-PAGE;
IV. Visualization, quantitation and identification of proteins

For details and description of other methods used for the study of inner ear proteins by the team at Washington University,  such as immunohistochemistry, cloning, consult the  References at the end of this section.  When available, MEDLINE links are provided.

 

I. Tissue Preparation

The minute size and complex structure of the auditory and vestibular end organs pose a variety of methodological obstacles to the elucidation of their biochemical and molecular biologic properties. A technique was developed some 30 years ago and has stood the test [Thalmann, 1976]. The approach consists of an integrated sequence of preparatory steps designed to produce, in vitro, information which closely approximates in vivo conditions in the tissues.  The technique is optimal for arresting all chemical processes of the tissue,  and for the preservation of morphology, allowing dissection of discrete tissue elements, such as organ of Corti, its substructures and even single cells.The technique consists of the following steps:

1. Quick-freezing of temporal bones with liquid nitrogen cooled to its melting point (-150^oC).
2. Freeze-drying in toto at -40^oC.
3. Dissection of discrete structures and substructures at room temperature and low relative humidity (40% or less).
4. Weighing of samples below a few micrograms on quartz-fiber fish pole balances, and above on an electronic balance above.

 II. Fluid Sampling

The method used for sampling cochlear endolymph in guinea pig follows closely that developed by Konishi et al. [1978];
a small fenestra is made, exposing the spiral ligament in the first turn. A dual-barrelled pipette-electrode with a tip diameter of 10 um is filled with mineral oil, and the recording electrode with 150 mM KCl. The pipette is advanced through the spiral ligament and stria vascualaris until a positive potential of 80-90 mV is measured. Up to 0.8 ul of endolymph is aspirated, while the endolymphatic potential is monitored. When the endolymphatic potential shows a sudden drop, or decreases continuously by more than 10 mV samples are discarded. This procedure assures clean samples, as previously determined by electrolyte determination and amino acid ratios.

For sampling cochlear perilymph in guinea pig a small fenestra is drilled into scala vestibuli of the first cochlear turn, and 1.0 ul of perilymph is aspirated by means of an oil-filled glass micropipette. Samples are examined microscopically for the presence of blood, and discarded if erythrocytes are visible.

Human perilymph is collected by either of two procedures: 1. Aspiration through a sterilized glass micropipette (1 mm OD, pulled to a tapered tip, diamter approximately 30 um) attached to a flexible tubing to aboid tip penetration or breakage during surgery. The micropipette is subseuqntly sealed by briefly heating both ends by electrocautery. 2. Application of pledget of sterile compressed foam (Merocel) at the locus of collection to absorb the perilymph sample. The foam is dropped directly into a tube, capped, and frozen.

For collection of endolymph of the endolymphatic sac in the guinea pig, the sampling of fluid samples has not been possible to this date. A method was developed most recently whereby freeze-dried endolymph is collected; the technique has been validated. The procedure follows the outline under Tissue Preparation (above). Specifically, the exposed skull base with the jugular vein intact is rapidly frozen and subsequently the lateral skull region including the temporal bone is separated in the frozen state. Following trimming, the preparation is freeze-dried in toto. Microdissection in the freeze-dried state is carried out at room temperature. The structures covering or adjacent to the endolymphatic sac, including the jugular vein, can be neatly separated, and debris covering the intracranial portion of the sac removed. The operculum is carefully chipped away, exposing the intraosseous portion of the sac. Segments of the sac wall are cautiously elevated with microblades and removed with hairpoints, thus exposing the silvery white cast of dried endolymph of the sac. If surgical procedures prior to freezing were performed with adequate care, not the slightest pink discoloration is visible which would signify hemorrhage or artifactual admixture of blood.

III. 1D- and 2D-Polyacrylamide Gel Electrophoresis (PAGE)

1D-PAGE:

For denaturing SDS gels, samples are solubilized in 'sample buffer' containing 6M urea, 0.12M Tris-HCl, pH6.8m 0.4% SDS, and 0.01M DTT. The electrophoresis follows the classical technique described by Laemmli [1979].

For nondenaturing IEF gels (used for the study of oncomodulin), samples are dissolved in deionized water, followed by double-strength sample buffer containing 60% glycerol and 4% ampholytes, then centrifuged for 2 min at 10,000 x g. IEF is performed at 15 W constant power in an SE600 vertical slab unit at 15^oC. The gel contains 5.5% acrylamide, 0.5% piperazine di-acrylamide, 10% (v/v) glycerol, and ampholytes (3.7% pI 3-10; 1.88% pI 3-5; 0.37% pI 2.5-4). Acetic acid (0.02M and 0.02M NaOH servs as the anolyte and catholyte, respectively. After pre-running for 15 minutes, samples are loaded, and focusing is continued for 150 minutes.

2D-PAGE

1. Fluid samples are heated for 5 minutes at 100^o in a solution containing 1% SDS and 2,3% DTE. They are subsequently diluted to a final SDS concentration of 0.25% or lower with a 'mix' containing 8M urea, 4% CHAPS, 40mM Tris-base and 65 mM DTE.
2. Tissue samples are solubilized in the 'mix' only and are not heated. For nondenaturing experiments the samples are dissolved in deionized water, followed by double-strength sample buffer containing

2D-PAGE is carried out on an immobilized pH gradient (IPG , Pharmacia 3-10 NL) in the first dimension and using the DALT technique [Anderson, 1988] in the second dimension. The gel gradient is poured with the aid of a computerized gradient maker (Angelique, Large Scale Biology, Rockville, MD).

IV. Visualization, Quantitation and Identification of Proteins:

Depending on the type of experiment that follows electrophoresis, and on the sensitivity required, gels are visualized by:

• Coomassie staining by the classical technique.
• Silver staining [Oakley et al., 1980 and modified by Merril [1990] and adjusted for thickness and number of gels].
• Copper staining [Lee et al., 1987].
• Zn staining [Fernandez-Patron, et al., 1992].
• Immunoblotting (Western blot) [Towbin et al., 1979] using either alkaline phosphatase-conjugated anti IgG or IgM, and NBT/BCIP or horseradish peroxidase-conjugated IgG and chemiluminescence, and using either nitrocellulose membrane or PVDF (Immobilon-PSQ for amino acid sequencing, and Immobilon-CD for mass spectrometry).

Proteins are identified and quantified by:

• Use of antibodies and standard proteins.
• Amino acid sequencing.
• Comparison with gels from other databases and subtractive analysis using the Amersham ImageMaster 2D Elite and Database software.
• Mass spectrometry (MALDI).

V. References: Pivotal references are provided here only. Updated information can readily be obtained from protocols distributed by companies, such as Amersham Pharmacia and BioRad, and on line from, for instance, from Expasy Molecular Biology Server (http://www.expasy.ch). Protocols have to be adapted for type of tissue, tissue preparation, and objective of study.

WHENEVER AVAILABLE, MEDLINE LINKS ARE PROVIDED

Tissue Sampling

THALMANN R (1976) Quantitative histo-and cytochemistry of the ear. In "The Handbook of Auditory and Vestibular Research Methods. CA Smith and J A Vernon (Eds.); CC Thomas, Springfield, IL, Part      Biochemistry, pp. 359-419.
MEDLINE 5081743  THALMANN R, THALMANN I, COMEGYS TH (1972) Quantitative cytochemistry of the organ of Corti. Dissection, weight determination and analysis of single outer hair cells. Laryngoscope 82:2059-2078.
MEDLINE 4320416  THALMANN R, THALMANN I, COMEGYS TH (1970) Dissection and chemical analysis of substructures of the organ of Corti. Laryngoscope 80:1619-1645.

Fluid Sampling

KONISHI T, HAMRICK PE, WALSH PJ (1978) Ion transport in guinea pig acochlea. Acta Otolaryngol 86:22-34.
SALT AN, THALMANN R (1988) Cochlear fluid dynamics. In: Physiology of the Ear. AF Jahn, J Santos-Sacchi (Eds), Raven Press, NY, pp 341-357.
MEDLINE 8084635 THALMANN I, KOHUT RI, RYU J, COMEGYS TH, SENARITA M, THALMANN R (1994) Protein profile of human perilymph: In search of markers for the diagnosis of perilymph fistula and other inner ear disease. Otolaryngol Head Neck Surg 111:273-80.
 

1D and 2D Polyacrylamide Gel Electrophoresis

LAEMMLI UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
ANDERSON L (1988) Two-dimensional electrophoresis. Operation of the ISO-DALT system. Washington DC: Large Scale Biology Press.
BERKELMAN T, STEMSTEDT T (1998) 2-D electrophoresis. Using immobilized pH gradients. Principle and methods. Manual. Amersham Pharmacia Biotech, pp. 1-50.
BioRad Catalog (latest edition)
A full protocol is provided on line from Expasy Biological Server

Visualization

MEDLINE 388439 TOWBIN H, STAEHELIN T, GORDON J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some application. Proc Natl Acad Sci USA 76:4350-3.
MEDLINE 6161559 OAKLEY BR, KIRSCH DR, MORRIS NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Ann Clin Biochem 105:361-363.
MEDLINE 2449094 LEE C, LEVIN A, BRANTON D (1987) Copper staining: a five minute protein stain for sodium dodecyl-sulfatepolyacrylamide gels. Anal Biochem 166:308-312.
MERRILL CR (1990) Silver staining of proteins and DNA. Nature 343:779-80.
FERNANDEZ-PATRON C, CASTELLANOS-SERRA L, RODRIGUEZ P (1992) Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: Sensitive detection of unmodified proteins. BioTechniques 12:564-573.