Notes Jba
1. Pipet pullers usually have a two-step pulling mechanism in which the first step pulls the capillary over a short distance to thin the glass. The second step with lower heat separates the capillary. Increasing or decreasing the heat of the first step changes the shape of the electrode to either more sharp or blunt, while an increase or decrease of the second pulling step results in electrodes with smaller or wider tip diameters, respectively. The fabrication of patch pipets is based on trial...
Detergent Extraction of Heart Gap Junctions
1. NaHCO3 buffer 1.0 L of 1 mM NaHCO3, pH 8.2, 1 mM phenylmethylsulfonyl fluoride PMSF, added as a solid to solutions . 2. KI Na2S2O3 NaHCO3 solution 200 mL of 0.6 M KI, 6 mM Na2S2O3, 1 mM NaHCO3, pH 8.2, 1 mM PMSF. 3. KI Na2S2O3 Tris solution 70 mL of 0.6 M KI, 6 mM Na2S2O3, 5 mM Tris-HCl, pH 9.0, 1 mM PMSF. 4. Tris buffer 400 mL of 5 mM Tris-HCl, pH 10.0, 1 mM PMSF. 5. 25 mL of 49 sucrose, 0.3 deoxycholate in 5 mM Tris-HCl, pH 10.0 solid PMSF added to a final concentration of 1 mM. 6. 35 mL...
Correction for Series andor Membrane Resistance
To investigate in which respects Fj benefits from correction for Rs and Rm under experimental conditions, we performed experiments on equivalent resistive circuits in a dual voltage clamp setup, using resistors having resistance values in the range normally present in actual dual whole-cell voltage-clamp experiments. The command voltage protocol of alternately stepping cell A and cell B see Fig. 1C was applied to obtain estimates of Rm1 and m2 through 18 Rm1 Rs2 ' Vba ' 4b Kb ' 4a Rs1 ' 4a ' 4b...
References Mnd
1. Gimlich, R. L., Kumar, N. M., and Gilula, N. B. 1990 Differential regulation of the level of three gap junction RNA's in Xenopus embryos. J. Cell Biol. 11, 597-605. 2. Dash, P., Lotan, I., Knapp, M., Kandel, E., and Goelet, P. 1987 Selective elimination of mRNA's in vivo complementary oligonucleotides promote RNA degradation by RNaseH-like activity. Biochemistry 84, 7896-7900. 3. Trexler, E. B., Bennett, M. V. L., Bargiello, T. A., and Verselis, V. 1996 Voltage gating and permeation in a gap...
Alkali Extraction Protocol
Initial enrichment of gap junctions is accomplished by the partial purification of liver plasma membranes, following the procedure described by Hertzberg 7 . Subsequently, nonjunctional membranes are removed by soni-cation in the presence of alkali. Differential centrifugation and density gradient centrifugation finally yields purified gap junction plaques containing Cx26 and Cx32. About the same amount of gap junction proteins can be obtained with this alkali procedure about 1 mg 150 g of...
Velocity Sedimentation of Connexons in Sucrose Gradients 1
This procedure was adopted from studies of the assembly of the nicotinic acetylcholine receptor 10 . It takes advantage of the fact that a 5-20 w w linear sucrose gradient is essentially isokinetic that is, a particle moves in the gradient at a constant velocity such that the distance through which it sediments is directly proportional to its sedimentation coefficient. As expected for a monomer that has a predicted molecular mass of 43 kDa, unassembled Cx43 is recovered in the 5S region...
Chicken Embryos
The Pluronic gel antisense ODN approach is ideal for studying connexin roles during development of the chick embryo see Note 4 . 1. Fertilized eggs see Chapter 9 in this volume for more details on the manipulation of fertilized chick eggs should be incubated at 38 C and staged according to Hamburger and Hamilton stages 11 . On removal from the incubator eggs are rotated 10x to ensure that the embryo is floating free at the top of the egg. 2. Clean eggs with 70 alcohol and then make a small hole...
MembraneImpermeant Tracers
1. Lucifer Yellow CH mol wt 443, two negative charges this tracer has a high fluorescence efficiency, which ensures its detection in minute levels. It also binds to cell components after aldehyde fixation and photoactivation, a property exploited to identify the cell s containing the tracer after histological processing at both light and electron microscopy levels 9,10 . The tracer is usually injected as a 2-4 solution in either distilled water or 1 M LiCl, and can be stored for weeks in the...
Riboprobe Synthesis 1
We have chosen to work with riboprobes cRNA instead of DNA probes, because 1 they can be synthesized with high efficiency and 2 they form, by hybridization with endogenous transcripts, RNA-RNA hybrids that are more stable than DNA-RNA hybrids 25 . The hybridization process can thus be performed at high stringency, and, in addition, the unbound and nonspecifically bound riboprobe can be digested by RNase without affecting the RNA-RNA hybrids. The DNA fragment selected for the synthesis of the...
Prehybridization and Hybridization Day 1
To obtain reliable results it is recommended to use at least five embryos for each developmental stage investigated and for each probe. 1. Bleach the embryos for 1 h at RT with 100 ethanol-30 H2O2 5 1, v v . This treatment also inactivates the endogenous phosphatases. 2. Rehydrate through a decreasing series of ethanol solutions in PBT 75 , 50 , 25 for 5 min each , followed by three washes in PBT. 3. Permeabilize with 10 g mL of proteinase K PK in PBT for 15 min at RT see Note 13 . 4. Incubate...
Prehybridization and Hybridization Steps
These solutions are prepared from molecular biology grade products. 2. Proteinase K Sigma 10 g mL in DEPC-treated water. 3. Glycine solution 2 mg mL glycine in PBT. 4. Fixation solution B 0.2 Glutaraldehyde in fixation solution A. The fixation solution B is made fresh as required from both fixation solution A and an 8 glutaraldehyde stock solution. The 8 glutaraldehyde stock solution is made from a 70 glutaraldehyde solution Sigma diluted with DEPC-treated water, aliquoted, and stored at -20 C....
Making Retroviral DNA Constructs
Cloning of full-length connexin cDNA or connexin gene fragments into viral DNA vectors involves two steps. First the DNA insert is cloned into an adaptor plasmid and then into a viral vector. The adaptor plasmid called CLA12NCO was originally constructed by Hughes et al. 3 . This miniplasmid can convert virtually any DNA segment into a CM fragment suitable for insertion into the retroviral vector RCAS . The CLA12NCO plasmid consists of a polylinker region with multiple restriction enzyme sites...
WaxEmbedding and Sectioning of the Stained Embryos
Precise identification of labeled tissues and cells requires serial sectioning of the embryos and examination of sections. The detection of a significant signal in sections necessitates that the embryos have developed a strong signal because part of the signal is lost during the unwaxing procedure with xylene. The procedure in the following section describes embedding in embedding wax, but, alternatively, pure paraffin can also be used see Subheading 3.2.1.2. . One day before use embedding wax...
Dissection and Preparation of the Frozen Specimens
1. The Dewar flask filled with liquid nitrogen is placed in the fume cupboard the solid metal cylinder, comprising a small receptacle at the top, is placed inside. Propane from the bottle is dispensed into the inner receptacle with the regulator valve adjusted to give a slow flow to fill it completely with condensed propane. 2. The material to prepare the samples has to be dissected immediately after the animals are killed. Very small 0.25 mm3 and thin pieces of the biological material were...
Introduction Otp
The utility of Xenopus oocytes as a system for the expression and electrophysiological analysis of many ion channels has been well documented. This system offers several advantages. The oocytes are robust in terms of their handling and care needs. This robustness, and their size allow for ready microinjection of cRNA into the cells without compromise to their health, or the use of sophisticated equipment. Thus, functional analyses can be accomplished within hours to days, as compared to the...
Apparatus
1. For RNA probe analysis Minigel electrophoresis apparatus Mupid-2, Eurogentec SA or equivalent, spectrophotometer Perkin Elmer . 2. Dissection material Sterilized scissors heavy model, 14 cm long, two sharp blades Barraquer Wolff, sharp and delicate blades, 5 mm long and tweezers of different sizes Moria, 12.5 cm long, serrated jaws Moria no. 5 straight extra delicate Dumont-Moria, straight, tips closing flat on 4 mm , sterile Petri-dishes 35 x 10 mm, Falcon , a binocular MZ6, Leica-Wild...
WholeCell vs Perforated Patch
As mentioned previously, the whole-cell patch-clamp recording mode brings the cytoplasm in continuity with the IPS. This is advantageous if one wants to change the composition of the cell interior see Table 1 . However, if one wants to study the effect of for example, extracellular signals on the behavior of gap junction channels via intracellular processes, the disruption of the cell machinery might largely interfere with the experiment. The advantage of the perforated patch method is that it...
NGlycosylation Tagging
Probably the most convincing method to determine the integration of a protein into the membrane bilayer, which in addition allows determination of the overall transmembrane topology of a membrane protein, is to introduce core glycosylation sites into the polypeptide sequence. This approach is based on the activity of oligosaccharyl transferase OST that is present in the lumen of the ER and within microsomes. The enzyme transfers mannose-rich carbohydrate moieties onto asparagine residues that...
References 1
1. Robertson, J. D. 1963 The occurrence of a subunit pattern in the unit membrane of club endings in Mauthner cell synapses in goldfish brain. J. Cell Biol. 19,201-221. 2. Benedetti, E. L. and Emmelot, P. 1965 Electron microscopic observations on negatively stained plasma membranes isolated from rat liver. J. Cell Biol. 26, 166-174. 3. Revel, J. P. and Karnovsky, M. J. 1967 Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 33, C7. 4. Kumar, N. M....
Equipment
1. Falcon INTEGRID 15 x 25 cm tissue culture dishes polystyrene, nonpyrogenic treated by vacuum gas plasma to optimize cell adherence . 2. Falcon Blue Max Polypropylene Conical Tubes, 15 and 50 mL Becton Dickinson . 3. Sterile glass bottles, 250 mL Pyrex . 4. Fisher disposable serological sterile pipets 2, 10, and 25 mL . 5. Fisher disposable Pasteur pipets, stored in Bellco glass cylinder and sterilized. 6. Plastic columns for chromatography of Tween-20 Econo-Pac 1.5 x 12 cm polypropylene...
Lipofection Procedure
For transfection of HeLa cells, we use the lipofection reagent Tfx-20 Promega, Madison, WI, USA see Note 7 . 1. HeLa cells and plasmid DNA are prepared as described previously for the calcium phosphate protocol see Subheading 3.2.1., steps 1-2 . 2. To 6 mL of standard medium in a sterile conical tube 13 mL , add 10 g of nonlinearized plasmid-DNA 1 g L in sterile H2O see Note 5 and vortex-mix the solution for 20 s. Afterwards, add the Tfx-20 suspension at a ratio of 4 1 v v to DNA solution and...
Gap Junctions Composed of Cx32 and Cx26 from Spodoptera Transfected Cultured
Cx32 and Cx26 have been successfully expressed in the insect cell line Sf9 from Spodoptera 12,13 . This procedure and subsequent extraction and purification of gap junction plaques is described in Stauffer 13 . An alkali extraction procedure was used similar to the Hertzberg technique described previously 7 , but with fewer centrifugation steps. Gap junctions consisting of Cx32, Cx26, or a combination of the two have been successfully expressed and purified. These are the connexins that are...
Detergent Extraction Protocol
Gap junctions are isolated as double membrane plaques by membrane fractionation and purification techniques see Fig. 1 for a schematic of the procedure . The isolation protocol, modified from Fallon and Goodenough 5 , produces exceptionally high yields of gap junctions up to 1 mg 150 g of liver with relatively high crystallinity and little amorphous material collagen contamination is also low. The two detergents used are Sarkosyl and Brij 58. The following protocol is for livers obtained from...
Probe Detection
1. Photographic emulsion Ilford Nuclear Research Emulsion G-5 see Note 6 . 2. Glycerol-water Add 1.2 mL of glycerol to 59 mL of bidistilled water. Filter through a 0.22 m filter and store at 4 C. 3. 10 Potassium bromide KBr in autoclaved bidistilled water. 4. Amidol-developer Prepare a fresh solution just before developing. Dissolve 1.13 g of Amidol 4-hydroxy 1,3-phenylene diammonium dichloride Fluka in 200 mL bidistilled water, add 4.5 g of Na2SO3 and bring it to a final volume of 250 mL with...