Bio-Dot® Microfiltration Apparatus Instruction Manual Catalog Numbers 170-6545 170-6547 For technical service, call your local Bio-Rad office or, in the U.S., call 1-800-424-6723.
Table of Contents Page Section 1 Introduction ...................................................................................1 1.1 Specifications ............................................................................................1 Section 2 Special Handling Features............................................................2 2.1 2.2 Autoclaving ................................................................................................2 Chemical Stability ..........................
Section 1 Introduction The Bio-Dot microfiltration apparatus can be used for any application requiring rapid immobilization and screening of unfractionated or purified proteins, nucleic acids, or macromolecular complexes on membranes, such as nitrocellulose or Zeta-Probe® membrane. The Bio-Dot apparatus is provided as a complete unit, or as a modular addition to the Bio-Dot SF slot format microfiltration apparatus.
Section 2 Special Handling Features The Bio-Dot apparatus withstands autoclave temperatures for sterilization, as well as cleaning with alcohols, acids, and base solutions. 2.1 Autoclaving The Tygon tubing and flow valve cannot be autoclaved. All other components of the apparatus withstand the autoclave treatment. After repeated autoclaving (~25 cycl es), the silicone rubber gasket may need replacing.
Section 3 Bio-Dot Assembly 3.1 Assembly 1. Clean and dry the Bio-Dot apparatus and gasket prior to assembly. 2. Place the gasket support plate into position in the vacuum manifold. (There is only one way to slide the plate into the manifold.) 3. Place the sealing gasket on top of the gasket support plate. The guide pins on the vacuum manifold help align the 96 holes in the gasket over the 96 holes in the support plate. Visually inspect the gasket to make sure the holes are properly aligned.
4. Always use forceps or wear gloves when handling membranes. Prewet the nitrocellulose or Zeta-Probe® membrane by slowly sliding it at a 45° angle into wetting solution. Note: PVDF membrane is not recommended. Wet nitrocellulose in 6x sodium, sodium citrate (SSC) for nucleic acid applications, and in tris-buffered saline (TBS) for protein blotting. Wet Zeta-Probe membrane in distilled water. See Sections 9 and 10 for solution preparation.
Flow valve setting 1. The vacuum manifold is exposed to the vacuum source only. Use for applying vacuum to the Bio-Dot apparatus. Flow valve Setting 2. The manifold is exposed to air. Use for gravity filtration procedures. Flow valve Setting 3. The manifold is exposed to both air and the vacuum. Use this setting for gentle vacuum applications where the amount of vacuum is regulated by putting a finger over the port exposed to the atmosphere. Fig. 3.
5. Check the wells after sample has been applied to ensure that there are no air bubbles in the wells. Air bubbles will prevent the sample from binding to the membrane. Air bubbles may be removed by pipetting the liquid in the well up and down. 6. Proper positioning of the flow valve relative to the level of the apparatus is important for proper drainage.
Section 4 Protein Blotting 4.1 Immunoassay Procedure Detailed instructions, including a comprehensive troubleshooting guide, for performing immunoassays are included in the Immun-Blot® instruction manuals. 1. Assemble the Bio-Dot apparatus as described in Section 3. Prewet the membrane prior to placing it in the apparatus. Nitrocellulose membranes are prewetted in TBS; nylon membranes, such as the Zeta-Probe membrane, are prewetted in distilled water (see Section 9 for solution preparation).
6. Adjust the flow valve so that the vacuum chamber is exposed to air. Add 200–400 µl of the Tween, tris-buffered saline (TTBS) wash solution to each well. Adjust the flow valve to the vacuum position and pull the wash solution through the membrane. Disconnect the vacuum as soon as the wash solution has drained from all the sample wells. Repeat the wash step. If the membrane is to be removed from the apparatus prior to performing an immunoassay, remove it at this point. Otherwise, proceed to step 7.
4.2 Special Protein Blot Applications 1. Soluble enzyme substrate reactions and quantitations Perform an immunassay as described in Section 4.1. Prior to color development, disconnect the vacuum and close the flow valve. Add an equal volume of substrate solution to all wells. Visualize positive reactions and record. For quantitation, withdraw equal aliquots of the soluble substrate reactant from each well and transfer to a plastic disposable microplate.
Section 5 DNA Blotting This section gives protocols for DNA blotting. The alkaline blotting method, using Zeta-Probe® membrane, and the standard method for DNA blotting to nitrocellulose is described. 1. The target DNA must be denatured prior to application to the membrane. When using the Zeta-Probe membrane, denature the DNA sample by addition of NaOH and EDTA solution to final concentrations of 0.4 M NaOH, 10 mM EDTA. Heat the sample to 100°C for 10 minutes to ensure complete denaturation.
Section 6 RNA Blotting RNA must be denatured prior to application to Zeta-Probe® or nitrocellulose membranes to ensure optimal hybridization. Two protocols are presented for denaturing RNA samples. 6.1 Alkaline RNA Denaturation and Fixation 1. Always wear gloves when handling blotting membranes. Pre-wet the blotting membrane by placing it gently at a 45° angle into a tray of wetting solution.
4. Assemble the Bio-Dot apparatus according to the instructions in Section 3.1. Remember to apply the vacuum and then retighten the screws that hold the apparatus together. 5. Samples and wash solutions may be applied with a standard pipet or a Costar Octapette pipet. Apply the denatured RNA, and pull the sample through by passive filtration or by applying a gentle vacuum. Note: a method for applying gentle vacuum to the apparatus is to adjust the flow rate valve to setting 3.
Section 7 Hybridization Protocols for Nucleic Acids 7.1 Probe Recommendations The specific activity, concentration, size range, and purity of the probe all have an important effect on signal-to-noise ratio during hybridization.
The carrier DNA used with nitrocellulose must be denatured before adding it to the prehybridization solution. Heat the DNA at 100°C for 5 minutes and cool rapidly. 3. Seal the top of the bag and incubate. For DNA or RNA Bound to Zeta-Probe Membrane 5 minutes at 65°C For DNA Bound to Nitrocellulose 2–4 hours at 68°C For RNA Bound to Nitrocellulose 8–20 hours at 42°C Hybridization 1. Cut one corner of the plastic bag.
7.3 Hybridization Protocols for RNA Probes The following protocols are for RNA probes to DNA blots. Casey and Davidson (1977) contains protocols for RNA-RNA hybridizations. Prehybridization 1. Place the blotted membrane inside a heat sealable plastic bag. Seal three sides, leaving the top side open. 2. Pipet in the prehybridization solution: For DNA Bound to Zeta-Probe Membrane (Bio-Rad Laboratories 1987) 50% formamide 1.5x sodium, sodium phosphate, EDTA (SSPE) 1% SDS 0.
Washes 1. At the completion of hybridization, remove the membranes from their hybridization bags into 2x SSC. Rinse briefly, then wash them sequentially with agitation for 15 minutes at room temperature in the following solutions: a. b. c. 2x SSC/0.1% SDS 0.5x SSC/0.1% SDS 0.1x SSC/0.1% SDS 2. For DNA bound to nitrocellulose membranes, it may be necessary to include an RNase treatment in the wash. Membranes are treated with 20 µg/ml RNase for 30 minutes at 37°C in 2x SSC. (Santzen et al. 1986) 3.
Section 8 Solutions for Protein Applications 8.1 Solutions for Nitrocellulose Membrane Tris Buffered Saline, 1x TBS, 2 L 20 mM Tris-HCl, pH 7.5 500 mM NaCl Dissolve 4.84 g Tris, 58.48 g NaCl in ~1.5 L distilled, deionized H2O. Adjust to pH 7.5 with HCl. Adjust the volume to 2 L with ddH2O. Tween 20 Wash Solution, 1x TTBS, 1 L 20 mM Tris, pH 7.5 500 mM NaCl 0.05% Tween 20 Add 0.5 ml Tween 20 to 1 L of TBS. Blocking Solution, 100 ml 1% BSA-TBS Add 1.0 g bovine serum albumin (BSA) to 100 ml TBS.
8.2 Solutions for Zeta-Probe Membrane for Protein Applications When immobilizing antigen onto the Zeta-Probe® membrane, the immunoassay must be performed in a separate container following removal of the membrane from the Bio-Dot apparatus. Two methods of blocking are given: Method A uses nonfat dry milk (Jerome and Jiehning 1986, Johnson et al. 1984) as the blocking agent. Method B uses gelatin and 1-methyl-2-pyrrolidinone (MPO) as the blocking agents.
Section 9 Solutions for Nucleic Acid Applications 20x SSC 3 M NaCl 0.3 M trisodium citrate (FW = 294.1) Dissolve 175.0 g NaCl and 88.2 g trisodium citrate in ddH2O. Adjust volume to 1 L with ddH2O. 20x SSPE 3.6 M NaCl 0.2 M Na2HPO4•7H2O 0.02 M EDTA Dissolve 210.0 g NaCl, 53.6 g Na2HPO4•7H2O, 7.44 g EDTA ddH2O. Adjust volume to 1 L with ddH2O. TE 10 mM Tris-HCl, pH 8.0 1 mM EDTA, pH 8.0 Dilute 10 ml 1 M Tris-HCl, pH 8.0 and 4 ml 0.250 M EDTA, pH 8.0 to 1 L with ddH2O.
50% Formamide Dilute 50.0 g formamide to 100 ml with ddH2O. Store at 4°C. Immediately before use, deionize the required volume by stirring gently for 1 hour with 1 g mixed-bed ion exchange resin (AG® 501-X8 (D) resin, catalog number 142-6425) per 10 ml of formamide. Filter through coarse filter paper. For DNA or RNA Bound to Zeta-Probe Membrane (Bio-Rad Laboratories 1987) 1 mM EDTA 7% SDS 0.5 M NaHPO4, pH 7.2 For DNA Bound to Nitrocellulose (Maniatis et al. 1982) 6x SSC 0.
Section 10 Troubleshooting Guide I. Filtration Apparatus 1. Leakage or Cross-Well Contamination a. Improper assembly. The screws must be retightened under vacuum following the initial assembly. b. Membrane is not properly rehydrated after assembly. Always rehydrate the membrane prior to applying samples. Apply vacuum only until solutions are removed from the sample wells, then disconnect the vacuum source. 2. No Filtration or Uneven Filtration Occurring a.
d. Proteins 15,000 daltons may show decreased binding to 0.45 µm nitrocellulose. Use the Zeta-Probe membrane or 0.2 µm nitrocellulose. Also, glutaraldehyde fixation will increase retention of small proteins and peptides to both nitrocellulose and the Zeta-Probe membrane. f. Protein may be removed from nitrocellulose by SDS, NP-40, or Triton X-100. Use Tween 20 in washes. Reduce concentrations or time of any SDS or NP-40 washes. III. High Background After Incubation With Labeled Probes 1. DNA and RNA a.
IV. Poor Detection Sensitivity or No Reactivity 1. DNA/RNA a. This problem may occur when total genomic DNA is probed for single copy or low copy number genes. Try the Zeta-Probe membrane for binding and retention of increased quantities of DNA. b. Hybridization was insufficient. Incorporate 10% dextran sulfate in the hybridization mixture. This polymer effectively reduces the solvent volume, thereby increasing the concentration of the solutes and enhancing hybridization. c. Exposure time was insufficient.
Section 11 Applications and References 12.1 Common Applications Protein Radioimmunoassay (RIA), enzyme-linked immunoassay (EIA), fluoroimmunoassay (FIA) of soluble or particulate cellular antigens (Cleveland et al. 1981, Shen et al. 1980) Analysis of enzymes (Faulstich et al. 1974, Huet et al. 1982) Hormone-receptor assays (Gershoni and Palade 1983, Schafer et al. 1974) Immunoglobulin detection (Herbrink et al. 1982, Wang et al. 1980) Hybridoma screening (Bennett and Yeoman 1983, Hawkes et al.
Bio-Rad Laboratories, Zeta-Probe Instruction Manual Brandsma J and Miller G, Nucleic acid spot hybridization: rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA, Proc Natl Acad Sci USA 77, 6851–6855 (1980) Bresser J and Gillespie D, Quantitative binding of covalently closed circular DNA to nitrocellulose in NaI, Anal Biochem 129, 357–364 (1983) Casey J and Davidson N, Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide, N
Locker D, Motta G, Detection of antibody secreting hybridomas with diazobenzyloxymethyl paper: an easy, sensitive and versatile assay, J Immunol Methods 59, 269–275 (1983) Lye DJ and Birge EA, The use of nitrocellulose filters to study DNA binding proteins in crude cell lysates: Effect of competing DNA, Curr Microbiol 6, 139–143 (1981) Maniatis T et al., Molecular Cloning: A Laboratory Manual, 1st edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982) Nakamura K et al.
Section 12 Ordering Information Catalog # Description 170–6545 Bio-Dot Microfiltration Apparatus, includes Bio-Dot sample template, vacuum manifold base plate, membrane support and gasket 170–6547 Bio-Dot Module, includes Bio-Dot sample template, membrane support and gasket 170–6546 Bio-Dot Gaskets, 3 gaskets per package 170–6542 Bio-Dot SF Microfiltration Apparatus, includes Bio-Dot SF sample template, vacuum manifold base plate, membrane support, gasket, and filter paper 170–6543 Bio-Dot SF Mod
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