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  Section: Biotechnology Methods » Molecular Biology
 
 
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Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method
  1. Cut PVDF membrane to the appropriate size, activate with absolute methanol for 5 sec, and incubate in distilled water for 5 min. For electroblotting, equilibrate in transfer buffer and follow the standard blotting procedure to transfer the proteins to the membrane. For dot blotting, keep the membrane wet until ready to use.
  2. After protein has been transferred to the membrane, wash again in absolute methanol for a few seconds and allow to dry at room temperature for 30 min or more.
  3. Block in 30 mL of 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), gently rocking for 1 hr at room temperature.
  4. Add appropriate dilution of primary antibody (typically 1:5000 or 1:10,000) prepared in 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), incubate 30 min at room temperature, gently rocking. Wash 3 times in 20 mL 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block) for 5 min each. Add appropriate dilution of secondary antibody conjugated to alkaline phosphatase prepared in 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), gently rocking for 30 min at room temperature.
  5. Wash as in step 5. Additionally, wash twice with 1X Western buffer without I-block.
  6. At the end of the second final wash, leave some buffer in the container to keep the membrane moist. With the membrane facing protein side-up, add 0.5 mL of substrate solution directly into the remaining liquid, mix well, and pipette (with a p10000) the solution over the membrane to ensure the entire surface comes into contact with the substrate. Gently agitate for a few minutes, remove the membrane to a paper towel, and let it dry completely. The substrate solution can be reused immediately for additional membranes.
  7. Scan membrane using the molecular dynamics storm.

Western Blotting Solutions
  • 10X Transfer buffer: 240 mM Tris, pH = 8.0; 1.92 M Glycine. Use 0.5X for
    transfer in 20% methanol.
  • 10X Western buffer: 200 mM Tris pH = 7.5; 1.5 M NaCl. To prepare 1X Western Buffer, dilute 10X buffer to 1X, adding Tween-20 to 0.1%. Remove 50 mL and set aside for the last 2 washes. To the remainder, add I-Block to 0.2%, heating gently with constant stirring until dissolved. Bring to room temperature before using (containing 0.1% Tween-20 and 0.2% I-Block).
  • Primary antibody: For his tagged proteins - Anti-His monoclonal antibody.
  • Secondary antibody: Goat anti-mouse alkaline phosphatase conjugated - Biorad #170-6520.
  • Substrate: ECF chemifluorescent substrate—Mix substrate with accompanying buffer as per manufacturer’s recommended instructions, prepare 1-mL aliquots, and store at –20°;C.

 
     
 
 
     




     
 
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