General Remarks About Strategies to Generate Antibodies Monoclonal Versus
Two strategies can be employed to obtain site-directed antibodies a polyclonal antibodies specific for a certain polypeptide region of the component in question and b monoclonal antibodies 4 specific for a particular epitope of functional importance. There are advantages and disadvantages for both of these types of antibodies. While a monoclonal antibody is specific for a single epitope, generating a specific monoclonal can be very time consuming one should estimate between 3 mon and 1 yr . On...
Indirect Immunofluorescence Analysis
2. Fix the cells by incubation in cold methanol acetone for 10 min at -20 C alternative protocols for fixation can be followed . 3. Wash with PBS to rehydrate. Under these conditions the coverslips can be stored up to a week at 4 C. For longer storage do not rehydrate and store dry at -20 C up to two months . 4. Wash the coverslips with PBS-T. Wipe out the excess of liquid. 5. Put the coverslips, cells face up, onto a parafilm foil in a humid chamber. 6. Add the appropriate dilution of primary...
Southern Blotting see Notes 6 and 7
1. Separate individual clones of the 180-bp dynein fragments from the pUC118 by digestion with BamHI and EcoRI, then gel-purify. 2. Denature DNA by boiling for 5 min, then immediately plunge into wet ice. 3. Label DNA with 32P dTTP using random primers DNA labelling system. 1. Digest 10 g genomic DNA with the appropriate enzyme. In the example shown in Fig. 3 see Note 7 , we used EcoRI. Perform the digestion in a large 100 L volume overnight. After digestion, precipitate the DNA with ethanol...
Materials 21 Cell Lines
In order to perform cell hybrid experiments, the different cells to be used should be selected according to their distinct keratin expression pattern, so they differ at least in the two polypeptides to be simultaneously analyzed. Usually a simple epithelium derived cell line can be used. However, other Fig. 1. opposite page Example of the dynamics of vimentin A, B, C,D versus keratin K6 A', B', D' or K5 C' upon the fusion between PtK2 simple epithelial cells, which also express vimentin and...
Production of Monoclonal Antibodies see Note 1
3.3.1. Injection Protocol 4 Wk Protocol 1. Day 0 1st immunization mix dissolved antigen with Freund's complete adjuvant at a ratio of 1 1 use about 100 g antigen per mouse see protocol for polyclonal antibodies . 2. Day 14 2nd injection for 2nd to 4th injection use antigen in Freund's incomplete adjuvant. 5. Day 30 collect some blood from the tail vein separate serum and use for immuno-test e.g., ELISA, immunoblot, dot-blot . 6. The desired titer of antibodies should be better than 1 500 for...
TatAntibody Coupling Chemical CrossLinking see Note 4
Dialyze antibody into coupling buffer or use a Centricon-30 Microconcentrator to exchange buffer. Adjust antibody concentration to 3 mg mL Freshly prepare 100 mM sodium periodate and keep on ice in the dark. Add 0.1 mL sodium periodate to 1 mL of purified antibody 3 mg total and incubate on ice for 20 min in the dark. Stop oxidation by adding 20 L of 1M glycerol and incubate on ice for 5 min. Dialyze overnight against coupling buffer. Determine volume and transfer sample to a 1.5 mL reaction...
Uffe Koppelhus Per HellungLarsen and Vagn Leick 1 Introduction
Rapidly swimming cells, like the ciliated protozoa, offer an interesting opportunity to study the chemosensory behaviour involved in chemo-attraction repulsion in response to external chemical stimuli. The membrane ultrastructure of ciliates resembles that of the chemosensory neurones and the olfactory epithelium in mammals 1 . As in higher organisms, the cilia may have adapted a sensory role. Ciliates may therefore be viewed as swimming receptors where correlations between cellular behaviour,...
HighResolution Light Microscope with Optics for DC and Fluorescence
The basic component of a high-resolution video microscope system is a research quality microscope Fig. 1 such as the Zeiss Axioplan with DIC and epi-fluorescence optics. The microscope must be equipped with a highresolution video camera e.g., Hamamatsu Newvicon tube camera and with an image processor e.g., Hamamatsu Argus-20 real-time image processor . Optional components include a low light-level camera for imaging fluorescent specimens e.g., Hamamatsu ORCA-2 dual scan cooled digital...
Use of GFP to Study Cytoskeletal Filaments
GFP-actin has been expressed in the eukaryotic microorganism Dictyostelium discoideum. In vitro studies demonstrated that purified GFP-actin was able to co-polymerize with native actin from D. discoideum to form filaments. These actin filaments were found to be competent substrates for myosin an actin-based motor discussed later in this chapter suggesting that GFP-actin does not compromise the functional integrity of actin filaments 8 . Further, in living cells expressing GFP-actin, functional...
Preparation of Holey Formvar Film
1. 0.5 w v Formvar in chloroform freshly prepared . 4. 50 mL Schott bottle wide neck . 6. Glass dish spray-painted black of 7 cm depth and 12 cm width or larger . From Methods in Molecular Biology, vol. 161 Cytoskeleton Methods and Protocols Edited by R. H. Gavin Humana Press Inc., Totowa, NJ Glass Petri dishes. Lens paper, lint-free. Magnetic stirrer. Methanol. Microprobe sonicator. Microscope slides. Razor blades. Staining jar.
Preparation of Holey Formvar Film and Holey Carbon Film see Note 2
1. Prepare fresh Formvar solution. Stir with magnetic stirrer for about 15-20 min. 2. Prepare fresh glycerol solution. 3. Add 15-20 drops of glycerol solution to dissolved Formvar. 5. Sonicate for 2 min at full power using a microprobe sonicator. Probe 1 3 from bottom of solution in a 50 mL Schott bottle see Note 3 . 6. Transfer solution into a staining jar. 7. Clean several microscope slides with lint-free lens paper. 8. Dip slides one at a time into the Formvar solution, remove rapidly and...
Immunofluorescence Reagents
1. Dulbecco's PBS D-PBS 8.1 mM Na2HPO4, 1.2 mM KH2PO4,138 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl2,0.5 mM MgCl2. 2. Pipes Buffer 0.1M Pipes pH 6.95 , 3 mM MgSO4,1 mM EGTA. 3. Blocking Buffer 5 normal goat serum, 5 glycerol in D-PBS, 0.04 sodium azide. 4. Triton-DPBS 0.1 Triton X-100 in D-PBS. 5. Formaldehyde-pipes fixative 2.5 formaldehyde in Pipes buffer. 6. Antifade mounting reagent such as ProLong or Slow-Fade light Molecular Probes, Inc., Eugene, OR .
Visualization of FActin Aggreates After Jasplakinolide Treatment
3.2.1. Chemical Fixation Electron Microscopy The procedure described herein was first used for Micrasterias cells by 1. Fix all cells, both treated samples and controls, in cacodylate-glutaraldehyde for 15 min see Note 3 . 2. Wash cells 3 times 5 min each in cacodylate buffer. 3. Postfix cells in cacodylate-osmium for 2 h. 4. Wash cells, as in step 2, 3 times 5 min each with distilled water. 5. Incubate cells in uranyl acetate for 2 h see Note 4 . 6. Wash 3 times 5 min each with distilled...