Electrophoretic Transfer in a Tank System
1. During gel electrophoresis, boil the nitrocellulose, and prepare the cold transfer buffer as described in Subheading 2. 2. Transfer the proteins from gel to nitrocellulose immediately on completion of SDS-PAGE to avoid leaching of protein from the gel. 3. Place the black side cathode of the plastic sandwich transfer apparatus on the tabletop use Fig. 1 as a guide . 4. Wet the Scotch-Brite pad in transfer buffer, and place over the sandwich. Wet three pieces of 3MM filter papers, and place...
for Preembedding Immunoelectron Microscopy
1. Deeply anesthetize rat with pentobarbital 100 mg kg, ip see Note 25 . 2. Surgically expose the heart and aorta. 3. Insert cannula see Subheading 2.1.1. into left ventricle, and gently push the can-nula until the tip enters the ascending aorta. Clamp cannula in place with hemostat. 4. Make a small excision in the right atrium, and begin perfusing immediately with 50 mL buffered saline containing heparin 1000 U mL followed immediately by 55 mL 2 paraformaldehyde 3.75 acrolein at a flow rate of...
Electrophysiological Recording Techniques
1. Ionic currents in isolated cultured epithelial cells are studied using whole-cell, tight-seal, patch-clamp recording methods 2 . 2. For electrophysiological recording, cells attached to glass cover slips are placed in a shallow chamber and positioned on the stage of a Nikon inverted microscope Fig. 3 . 3. The chamber is superfused with standard external solution from a series of reservoirs and valves designed to provide a regulated gravity-fed flow rate of 1-2 mL min. Most drugs and ligands...
Ligation of PCR Fragments into pBluescript
1. Prepare a ligation reaction for each isolated and prepared PCR band, in addition to one control that contains everything except insert DNA this control will determine the number of background colonies following transformation. 2. To 1 L of cut-vector stock or amount that yields satisfactory background see Note 17 , add 5-10 L of digested PCR DNA to approx 1 3 molar ratio , 2 L of 10X ligation buffer supplied with enzyme , 2 L of ligase, and sterile water to 20 L final volume. 3. Mix and...
Preparation of Cytosolic S130 and Polysome Fractions
These preparations were originally described by Brewer and Ross 44 and are a refinement beyond S100 preparations in that the concentration of RNA binding proteins is increased. However, this is coincident with increased RNase activities. 1. Cells are washed twice with cold, serum-free medium DMEM, and resuspended in 3.5 mL of buffer A 10 mM Tris-HCl, pH 7.6, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM DTT . 2. Cells 2 x 108 are homogenized with a tightly fitting Dounce homogenizer 20...
Identification of Adrenergic Receptor Modulation of IKCa in PCE Cells
K Ca channels are targets for modulation by a number of agonists 20 . Adrenergic regulation of IK Ca in PCE cells is examined by recording whole-cell currents in standard recording solutions before and after pneumatic pressure application of the nonselective adrenergic receptor agonist EPI or the selective arAR agonist, PHe. 1. Application of 100 M EPI for 40 s from a puffer pipet increases the whole-cell outward current measured at 58 mV by 117 Fig. 5A . To confirm that EPI is increasing the...
DNA Binding Proteins and Electrophoretic Mobility Shift Assay EMSA
DNA binding proteins are involved in the regulation of a variety of molecular mechanisms, including transcriptional activation 14 . Tissue-specific transactivating proteins can bind to cis-acting DNA sequences or hormonal response elements HREs to regulate gene transcription. The EMSA is commonly utilized to investigate the interactions between a target sequence DNA or RNA and cellular proteins Fig. 1 15-17 . EMSA is a simple and rapid technique used to detect nuclear or cytoplasmic proteins...
Saturation Binding
Subtype analysis by competition binding is suitable as long as the radioligand itself is not highly subtype-selective. Whereas both 125ICYP and 3H CGP-12177 exhibit little selectivity for P1-AR and P2-AR, they both have a much lower affinity for P3-AR as do the antagonists alprenolol and propranolol. To discriminate between P3-AR and P1-AR, we use a saturation binding assay that has been modified from our standard assay as follows the volume is kept at 250 L, the concentration of 125ICYP is...
Preparation and Electrophoresis of Sequencing Gel
A number of sequencing apparatus are commercially available. The method described below for pouring and conducting electrophoresis of a sequencing gel is optimized for the Bio-Rad Sequi-Gen sequencing apparatus, which includes a detailed set of instructions. 1. Thoroughly wash gel plates with glass cleaner and then with 100 ETOH. To ease removal of the outside plate after electrophoresis of the gel, coat the gel-facing side of this plate with a solution of 2 silane in chloroform toxic wear...
The Eukaryotic Vectors
A large variety of vectors for expression in eukaryotic cells are described in the literature. The different types of plasmids that have been used to express adrenergic receptor in eukaryotic cells can be classified into three groups, specifically monogenic, bigenic, and bicistronic vectors. The general characteristics of these plasmids are depicted in the Fig. 1. Monogenic vectors are the simplest form of expression plasmids. Briefly, they consist of a single transcription unit containing a...
Capillary Transfer of RNA to Solid Support
1. Cut nylon membrane and 4 Whatman 3 MM filters to the same size as the gel. 2. Set up gel capillary transfer apparatus i.e., the sandwich box as follows see Note 11 Fill reservoir with 20X SSC place wick i.e., the sponge on a platform suspended above reservoir and submerge both ends of the wick in reservoir prewet four Whatman filter papers in 20X SSC, and place two of them on top of the wick place gel on top of the Whatman filter papers with the open side of the wells facing down place nylon...
Collection and Preparation of Preimmune Rabbit Serum
1. Prior to immunizing the rabbits, preimmune serum is collected from each rabbit to neutralize nonspecific immunoreactive proteins. 2. Blood is collected from the ear vein of the rabbit. 3. Dilate the ear vein by swiping the external region of each ear with a xylene-saturated gauze. 4. Remove the red top cap from the Vacutainer tube. 5. After dilation, insert the needle into the vein using the butterfly handle as a guide. 6. When blood begins to flow in the catheter, connect its end into the...
Notes Xji
1. The supernatant is radioactive and should be discarded in accordance with radioactive waste disposal procedures. 2. AA is highly sensitive to oxidation across its multiple double bonds. It is also light-sensitive. Store under nitrogen or argon in an opaque container, and avoid direct exposure to sun or fluorescent light. 3. Aerate Krebs' buffer with 95 O2 5 CO2 at least every 10 min during washout and experiment. Also, aerate test tubes 1 min prior to placing uptake probes into them. Airflow...
Storage of Fixed Brain Sections
1. 24-Well plastic tissue-culture dishes. 2. Small glass vials with lids e.g., 22-mL scintillation vials . 3. Cryoprotectant solution 38 Combine 500 mL 0.1 M sodium phosphate buffer 500 mL dH2O, 1.59 g NaH2PO4 H2O, 5.47 g Na2HPO4 , 300 mL ethylene glycol Sigma , 300 g sucrose, 10 g polyvinylpyrolidone PVP-40 Sigma . Stir to dissolve it is difficult to get PVP into solution . Adjust volume to 1000 mL with dH2O. Store at 4 C.
DNase I Footprinting Assay
1. Binding buffer 25 mM HEPES, pH 7.6, 40 mM KCl, 0.1 mM ethylenediamine tetra-acetic acid EDTA , 1 mM dithiothreitol DTT , 10 glycerol. 3. Ca2 Mg2 solution 5 mM CaCl2, 10 mM MgCl2. 4. DNase I stop solution 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0, 2 sodium dodecyl sulfate SDS , 0.1 mg mL proteinase K note the proteinase K must be added just prior to use . 5. Proteinase K Prepare stock solution 100 mg mL , and store at -20 C. 6. RQ1 RNase-free DNase stock from Promega Madison, WI store at -20...
Amplification and Receptor Production
1. Using Sf9 cells grown to 80 confluence in a 170-cm2 culture flask, add 0.5 mL suspension of a selected virus clone into 50 mL of culture medium. Allow the infection to develop for 6 d at 27 C then collect the culture medium. Pellet the cell debris by centrifugation 3000g 10 min . The resulting supernatant constitutes the preamplification supernatant used in subsequent steps. 2. In a culture spinner containing 250 mL culture medium supplemented with 0.1 pluronic acid , infect an insect cell...
Superfusion Assay of [3HAA Release
The method described here measures the release of 3H AA following incorporation into cellular phospholipids by using a superfusion system with BSA as a carrier. The use of a superfusion system eliminates each of the limitations of the static incubation method described above. The continuous flow of medium minimizes reuptake or metabolism of liberated AA. The cells are sealed in a Teflon chamber, removing the influence of pipeting and medium changes. Responses are still variable, but with...
Preparation of Nuclear and Cytoplasmic Extract from Tissues or from Cultured
2.1.1. The Extraction of Nuclear and Cytoplasmic Proteins from Suspension and Adherent Cell Lines 1. Monolayer adherent mammalian cells e.g., HeLa, CV-1 or suspension e.g., Sup-T1 cultures see Note 5 . a. 1X Phosphate-buffered saline PBS . e. 1M Dithiothreitol DTT stored at -20 C. f. 0.1 M Phenylmethylsulfonyl fluoride PMSF Solvent is 100 isopropanol. Store at 4 C. g. 1000X Protease inhibitor cocktail 1 mg mL aprotinin, 0.5 mg mL Leupeptin, 0.7 mg mL pepstatin . Store at -20 C. 3. Buffer A 10...
Conjugation of MBS to KLH
The first step in this procedure is to determine the solubility of your synthetic peptide and to prepare the Sephadex G-25 column. 1. Test the solubility of the peptide in 20 mM EDTA, 0.1 M sodium phosphate, pH 6.8. If the peptide is not soluble in this buffer, try the buffers described in Subheading 4. see Notes 1 and 2 . 2. Swell the Sephadex G-25 in 0.1 M sodium phosphate, pH 6.8, and load into the disposable polyethylene column. 3. Prior to coupling the peptide to KLH, the lysine side chain...
Description of the SixChannel Superfusion System
The methods were developed using the SF-06, a six-channel superfusion system from Brandel Gaithersburg, MD . Identical methods can be used with the SF-12 or 12-channel system. The system is illustrated in Fig. 3 using a Fig. 3. Schematic of the Brandel superfusion system. For the sake of simplicity, only a single channel is depicted. Fig. 3. Schematic of the Brandel superfusion system. For the sake of simplicity, only a single channel is depicted. single channel for the sake of simplicity. A...
Solutions
1. Ammonium acetate 10 M , DEPC-treated and autoclaved. 20X SSC 0.3 M sodium citrate, pH approx 7.0, containing 3 M NaCl. 2X SSC 1 10 dilution v v of 20X SSC concentrate in DEPC-treated water. 1X SSC 1 20 dilution v v of 20X SSC concentrate in DEPC-treated water. 3. Box buffer Volume required depends on the size of container used. Made by mixing equal volumes of 4. DEPC-treated water 5 L. Prepare 0.1 solution 1.0 mL DEPC L with highest- quality filtered water, stirring overnight vigorously...
Steven R Post Rennolds S Ostrom and Paul A Insel 1 Introduction
Many drugs and hormones interact with plasma membrane receptors to induce changes in the production of intracellular second messengers. The first such messenger to be identified was adenosine-3',5' cyclic monophosphate cAMP , discovered by Sutherland and Rall in the late 1950s 1 . Changes in cellular cAMP levels can occur from increases or decreases in its biosynthesis which results from the catalytic conversion of ATP to cAMP by the enzyme adenylyl cyclase or by altering its degradation which...
Rapid Amplification of cDNA Ends RACE 1
After a product from RT-PCR of cDNA has been cloned and identified, that fragment can be used to screen a cDNA library in a similar manner to that described for screening a genomic library see Subheading 3.1. . Alternatively, the small amount of sequence information derived from the RT-PCR fragment can be used to amplify sequences from both the 3' end 3' RACE and 5' end 5' RACE of the target message. The cDNA used above to construct the library see Chapter 1, step 11 in Subheading 3.2.1. can be...
Preparation of Human Tissues see Notes 1 and 2
During tissue harvest ideally collected lt 2 h after death , cut tissue blocks into manageable pieces i.e., section size should permit slide-mounting without distortion . 1. To provide initial tissue fixation, immediately immerse tissue in ice-cold 4 paraformaldehyde solution 4 C in 50-mL disposable conical tubes 4-6 h, no more than 24 h . 2. To eliminate air and freezing artifact, rinse tissue in ice-cold 20 sucrose in PBS, and then immerse in fresh ice-cold 20 sucrose in PBS 5X tissue volume...
References Kon
1. Ruoho, A. E., Rashidbaigi, A., and Roeder, P. E. 1984 Approaches to the identification of receptors utilizing photoaffinity labeling, in Membranes, Detergents, and Receptor Solubilization, Liss, New York, pp. 119-160. 2. Ruoho, A. E., Kiefer, H., Roeder, P. E., and Singer, S. J. 1973 The mechanism of photoaffinity labeling. Proc. Natl. Acad. Sci. USA 70, 2567-2571. 3. Atlas, D. and Levitski, A. 1978 Tentative identification of beta-adrenoreceptor subunits. Nature Lond. 272, 370-373. 4....
Electrophoretic Blotting of Proteins
1. Apparatus, power supply, and reagents are similar to those used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE of proteins. 2. Prestained protein standard. 3. Nitrocellulose membranes and Whatman 3MM paper. 4. Western transfer buffer 48 mM Tris base, 39 mM glycine, 0.037 SDS, 20 methanol. Fig. 1. Identification of the factors binding to footprint II on the P2 promoter of the a1B-AR gene by Southwestern blotting. Nuclear extracts from rat brain or liver were...
Transcriptional Pulse
Transient promoter activation transcriptional pulse provides a potentially less toxic means to measure mRNA half-life. One method involves placing the gene of interest downstream of the serum-inducible c-fos promoter 41 . Cells expressing the gene, either transiently or stably, are incubated in low serum 0.5 to render them quiescent. Subsequently, the cells are exposed to 10-15 serum which rapidly and transiently 15-30 min induces the c-fos promoter. Following withdrawal of serum stimulus, the...
TimeCourse Experiments
Time-course experiments, in which the excitation and emission wavelength are fixed and fluorescence is measured over time, can be used as a highly sensitive method to identify ligand-induced conformational changes that lead to changes in the polarity of the environment surrounding the incorporated fluorophore 10 . As illustrated in Fig. 2, binding of the full agonist, isoproterenol, to the IANBD-labeled P2-AR causes a decrease in fluorescence reaching a maximum amplitude below the extrapolated...
DoubleLabeling Technique Using Peroxidase Substrates that Result in Reaction
3.2.3.1. Double Labeling Using DAB and NiDAB e.g., Labeling a2C-ARs and TH in Catecholaminergic Cells see Note 14 1. Follow steps 1-5 in Subheading 3.2.1. 2. Follow step 6 in Subheading 3.2.1. selecting for this incubation the primary antibody 1 that will be detected using the ABC method e.g., a2A-AR antibody see Note 15 . 3. Follow steps 7-11 in Subheading 3.2.1. select biotinylated secondary antibody directed against primary antibody used in step 2 . 4. Prepare peroxidase substrate just...
Detailed Description of Transcardial Perfusion for EM
Transcardial perfusion with fixatives is one of the most critical steps for successful ultrastructural preservation of tissue. The aim of transcardial perfusion is to achieve ultrastructural preservation before morphological and presumably chemical alterations of tissue are triggered by anoxia. In order to minimize artifactual alterations of tissue, anoxia may be minimized by maintaining artificial ventilation during transcardial perfusion. The other key to success is speed, i.e., minimizing...
Choice of Methods for Fixation of Tissue Transcardial Perfusion vs Immersion
For optimal preservation of cellular morphology and of the distribution of molecules within cells, transcardial perfusion with fixatives is required. However, there sometimes are needs to analyze the ultrastructure of tissue that has not undergone transcardial perfusion. One example is the need to analyze the ultrastructure of biopsy samples or blocks of tissue obtained postmortem. Even brains that have undergone transcardial perfusion with fixative may need to be postfixed by immersion for...
References Sqx
1. Robison, G. A., Butcher, R. W., and Sutherland, E. W. 1968 Cyclic AMP. Annu. Rev. Biochem. 37, 149-174. 2. Lands, A. M., Arnold, A., McAuliff, J. P., Luduena, F. P., and Braun, T. G. 1967 Differentiation of receptor systems activated by sympathomimetic amines. Nature 214, 597,598. 3. Arch, J. R. S. and Kaumann, A. J. 1993 p3 and atypical P-adrenoceptors. Med. Res. Rev. 13, 663-729. 4. Stadel, J. M. and Lefkowitz, R. J. 1991 Beta-adrenergic receptors identification and characterization by...
HEK 293 Membrane
1. HEK 293 cells stably transfected with the human recombinant 2-AR were scraped from plates using HME buffer 8 mL of buffer for 150-mm dish . 2. Homogenize the cells on ice with seven strokes in a Dounce homogenizer. 3. Prepare 15 mL SW 28.1 rotor tubes with 2.5 mL of 23 sucrose in HE buffer layered carefully over 2.5 mL 43 sucrose in HE buffer. 4. Layer lysates carefully on top of sucrose. 5. Centrifuge at 4 C for 40 min at 25,000 rpm in a Beckman SW 28.1 rotor. 6. Membranes are collected at...
Preparation of Nuclear and Cytoplasmic Extract from Tissues or from Cultured
The method utilized to isolate nuclear and cytoplasmic extracts employs the use of hypotonic lysis of the plasma membrane, followed by high-salt extraction of nuclei. 3.1.1. Extraction of Nuclear and Cytoplasmic Proteins from Adherent Cell Lines see Note 1 Note All centrifugations should be performed at 4 C with samples kept on ice at all times. It is preferable to conduct protein isolations in a cold room. 1. Rinse the cells briefly with ice-cold 1X PBS. Using a cell scraper, scrape cells into...
YiTang Tseng and James F Padbury 1 Introduction
There are several methods currently available for transfection of DNA into mammalian cells. These include transfection with calcium phosphate 1 , diethylaminoethyl DEAE -dextran, cationic liposomes, nonliposomal lipid compounds, and electroporation 2 . Each of these methods has advantages and disadvantages selection of transfection method is based on the cell types used and personal preference. Transfection using calcium phosphate is highly efficient and is the most cost-effective method...
Adrenergic Receptor Signal Transduction Pathways and AA Release
Adrenergic receptors are members of the large family of integral membrane proteins that are coupled to guanine nucleotide binding regulatory proteins or G-proteins. G-protein-coupled receptors are important physiological regulators, because they are the targets for a large number of hormones and neu-rotransmitters, as well as autocrine and paracrine factors. Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as endocrine and...
Hybridization Pxy
2. Hybridization buffer 20 mM PIPES, pH 7.0, 50 formamide, 2 mM EDTA, 0.8 M NaCl, 0.2 SDS, 0.02 Ficoll, 0.02 polyvinylpyrrolidone, 0.02 bovine serum albumin BSA , and 500 g mL denatured salmon sperm DNA. Make fresh before use. 3. 20X SSC 3 M NaCl, 0.3 M tri-sodium citrate, 6X SSC, 2X SSC, and 0.5X SSC made by dilution of 20X SSC.