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For a immunoprecipitation experiment: By Observe the pellets and supernatants under UV light. Which tube contains green fluorescent protein?

For a immunoprecipitation experiment: By Observe the pellets and supernatants under UV light. Which tube contains green fluorescent protein?

Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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For a immunoprecipitation experiment: By Observe the pellets and supernatants under UV light. Which tube contains green fluorescent protein?
In this practical, you will carry out a simple immunoprecipitation experiment to isolate a protein of interest. A fluorescent-tagged protein is used to make visualization of immunoprecipitation easier. Materials 30 μl cellular extract 30 μl BE antibody 1 30 μl BE antibody 3 1 vial of Resin (protein-A) 3 ml PBS 6 centrifuge tubes (1.5 ml) Procedure 1. Label 3 centrifuge tubes as IP-1, IP-2, and IP-3 with your group name. 2. 3. 4. 5. 6. 7. 8. 9. Vortex the Resin (Protein-A) tube briefly. Using a wide bore tip (cut the tip with a blade or a pair of scissors), pipette 15 µl Resin (Protein-A) suspension to each labeled tube. Wash the Resin (Protein-A): Pipette 0.1 ml PBS to each tube. Centrifuge for 5 seconds at 1,000 g. Carefully remove and discard the supernatant without disturbing the agarose beads. 1 Set up three tubes as in the following table: Tube PBS Cellular extract Antibody 1 Antibody 3 IP-1 IP-2 IP-3 280 μ. | 290 μ. | 280 με 10 μl 10 μl 10 μl 10 μl Close all tubes tightly. Incubate the tubes at 4°C for 60 min. Invert the tubes 3-4 times. every 10 min. Label 3 tubes for collecting the supernatants as "IP-1 SN", "IP-2 SN", and "IP-3 SN". Centrifuge all tubes 5 seconds at 3,000 rpm. Carefully transfer the supernatants to the labeled tubes without disturbing the agarose beads. Add 300 μl PBS to each tube. Vortex briefly or tap the tubes with fingers to suspend the agarose beads. Centrifuge all tubes 5 seconds at 3,000 rpm. Carefully remove and discard the supernatant without disturbing the agarose beads. 10. Repeat step 8 and step 9. 11. Observe the pellets and supernatants under a UV transilluminator. 2
Transcribed Image Text:In this practical, you will carry out a simple immunoprecipitation experiment to isolate a protein of interest. A fluorescent-tagged protein is used to make visualization of immunoprecipitation easier. Materials 30 μl cellular extract 30 μl BE antibody 1 30 μl BE antibody 3 1 vial of Resin (protein-A) 3 ml PBS 6 centrifuge tubes (1.5 ml) Procedure 1. Label 3 centrifuge tubes as IP-1, IP-2, and IP-3 with your group name. 2. 3. 4. 5. 6. 7. 8. 9. Vortex the Resin (Protein-A) tube briefly. Using a wide bore tip (cut the tip with a blade or a pair of scissors), pipette 15 µl Resin (Protein-A) suspension to each labeled tube. Wash the Resin (Protein-A): Pipette 0.1 ml PBS to each tube. Centrifuge for 5 seconds at 1,000 g. Carefully remove and discard the supernatant without disturbing the agarose beads. 1 Set up three tubes as in the following table: Tube PBS Cellular extract Antibody 1 Antibody 3 IP-1 IP-2 IP-3 280 μ. | 290 μ. | 280 με 10 μl 10 μl 10 μl 10 μl Close all tubes tightly. Incubate the tubes at 4°C for 60 min. Invert the tubes 3-4 times. every 10 min. Label 3 tubes for collecting the supernatants as "IP-1 SN", "IP-2 SN", and "IP-3 SN". Centrifuge all tubes 5 seconds at 3,000 rpm. Carefully transfer the supernatants to the labeled tubes without disturbing the agarose beads. Add 300 μl PBS to each tube. Vortex briefly or tap the tubes with fingers to suspend the agarose beads. Centrifuge all tubes 5 seconds at 3,000 rpm. Carefully remove and discard the supernatant without disturbing the agarose beads. 10. Repeat step 8 and step 9. 11. Observe the pellets and supernatants under a UV transilluminator. 2
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