407211 INSTA-Blot™, Human Tissues

407211
  
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      Overview

      Replacement Information

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      Description
      Overview

      This product has been discontinued.





      Useful for the detection of expressed proteins of interest from various human tissues. Contains ~20 µg per lane of the following tissue extracts: brain, heart, small intestine, kidney, skeletal muscle, stomach, spleen, ovary, and testis.
      Catalogue Number407211
      Brand Family Calbiochem®
      References
      ReferencesAusubel, F., et al. 1998. Current Protocols in Molecular Biology, Vol. 2, 10.8.1.
      Product Information
      FormMembrane containing immobilized proteins
      Applications
      Key Applications Immunoblotting (Western Blotting)
      Application NotesImmunoblotting (see comments)
      Application CommentsProtocol
      Note: This protocol is provided only as a guide. Variables associated with the individual assay conditions will dictate the optimal working dilutions.

      1. Soak the blot in 100% methanol until the blot is wet, followed by 2 washes with TBST (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.01% Tween®-20 detergent) to remove any residual methanol.
      2. Incubate the blot for 1 h with 2% nonfat dry milk in TBST to block non-specific binding.
      3. Incubate the blot with primary antibody in 2% milk/TBST for 1-2 h at room temperature or overnight at 4°C. Dilute the primary antibody according to the manufacturer's recommendations.
      4. Following incubation with the primary antibody, wash the blot 5 times in TBST and incubate with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Dilute the secondary antibody according to the manufacturer's recommendations.
      5. Wash 5 washes with TBST and develop the blot for ~5 min using a chemiluminescence substrate.
      6. Expose the blot to X-ray film for an appropriate time period (usually 10 s to 20 min) to visualize the protein bands.
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Ambient Temperature Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Protect from Light Protect from light
      Do not freeze Ok to freeze
      Special InstructionsFor short-term storage, keep at room temperature. For long-term storage freeze (-80°C) in the dark.
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      INSTA-Blot™, Human Tissues Certificates of Analysis

      TitleLot Number
      407211

      References

      Reference overview
      Ausubel, F., et al. 1998. Current Protocols in Molecular Biology, Vol. 2, 10.8.1.

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      Data Sheet

      Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

      Revision11-June-2009 JLN
      ApplicationImmunoblotting (see comments)
      DescriptionINSTA-Blot™ ready-made blots eliminate the preparation of tissue lysates and the running and transferring of SDS-PAGE gels. The INSTA-Blot™ Human Tissues contains approximately 20 µg per lane of different human tissues, resolved onto Immobilon membrane. The membrane is stained with amido black to permit quantitation of proteins prior to immunoblotting. The blot contains the following tissues:

      Lane 1. Molecular weight marker
      Lane 2. Brain
      Lane 3. Heart
      Lane 4. Small Intestine
      Lane 5. Kidney
      Lane 6. Liver
      Lane 7. Lung
      Lane 8. Skeletal Muscle
      Lane 9. Stomach
      Lane 10.Spleen
      Lane 11.Ovary
      Lane 12.Testis

      MW Markers: 180 kDa, 116 kDa, 97 kDa, 66 kDa, 48 kDa, 29 kDa, 18 kDa, and 14 kDa.
      BackgroundThe identification of the human genome has promoted novel studies involving aging, behavioral research, diabetes and obesity, immunology, bioassay and pharmacological screening. Immunoblot analysis assists scientists conducting such research to make important contributions to functional genomics, allowing for structural, functional, and comparative analysis of proteins in human models. Calbiochem® brand is pleased to introduce a ready-to-use membrane that provides a simple and fast solution for screening the expression of a particular protein in human tissue.
      FormMembrane containing immobilized proteins
      Recommended reaction conditionsINSTA-Blot™ Protocol: 1. Soak the blots in 100% methanol until the blot is wet, followed by washing with TBST (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.01% Tween 20®)-detergent twice to remove any residual methanol. 2. Incubate the blots for 1 h with 2% nonfat dry milk in TBST to block non-specific binding. 3. Incubate the blots with primary antibody in 2% milk/TBST for 1-2 h at room temperature or overnight at 4°C. The primary antibody can be either a purified monoclonal antibody or polyclonal antibody at a standard concentration of 1-2 μg/ml or as a hybridoma tissue culture supernatant diluted 1:5 with 2% milk/TBST. 4. After incubation with the primary antibody, wash the blots 5 times in TBST and incubate with a secondary antibody conjugated to horseradish peroxidase (~1:1000-1:3000 dilution) for 60 min at room temperature. 5. After 5 washes with TBST, develop the blots for 5 min usinga chemiluminescence substrate (for example, SuperSignal® HRP Substrate, Cat. No. 69059). 6. Expose the blots to X-ray film for an appropriate time period (usually 10 s to 20 min) to visualize the chemiluminescent signal corresponding to the specific antibody-antigen reaction. Note: This protocol is provided only as a guide. Variables associated with the individual assay conditions will dictate the optimal working dilutions. Colorimetric detection (either alkaline phosphatase or HRP-conjugated secondary antibody and an appropriate substrate) may also be used; however, chemiluminescent detection is more suitable for weakly expressed proteins.
      CommentsProtocol
      Note: This protocol is provided only as a guide. Variables associated with the individual assay conditions will dictate the optimal working dilutions.

      1. Soak the blot in 100% methanol until the blot is wet, followed by 2 washes with TBST (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.01% Tween®-20 detergent) to remove any residual methanol.
      2. Incubate the blot for 1 h with 2% nonfat dry milk in TBST to block non-specific binding.
      3. Incubate the blot with primary antibody in 2% milk/TBST for 1-2 h at room temperature or overnight at 4°C. Dilute the primary antibody according to the manufacturer's recommendations.
      4. Following incubation with the primary antibody, wash the blot 5 times in TBST and incubate with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Dilute the secondary antibody according to the manufacturer's recommendations.
      5. Wash 5 washes with TBST and develop the blot for ~5 min using a chemiluminescence substrate.
      6. Expose the blot to X-ray film for an appropriate time period (usually 10 s to 20 min) to visualize the protein bands.
      Storage +2°C to +8°C
      Protect from light
      Do Not Freeze Ok to freeze
      Special InstructionsFor short-term storage, keep at room temperature. For long-term storage freeze (-80°C) in the dark.
      Toxicity Standard Handling
      ReferencesAusubel, F., et al. 1998. Current Protocols in Molecular Biology, Vol. 2, 10.8.1.