PRE-LAB 7 Western Blot of RuBisCo Pre-laboratory Assignment Directions: TYPE the

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PRE-LAB 7 Western Blot of RuBisCo Pre-laboratory Assignment Directions: TYPE the answers to the following questions and turn in BEFORE lab begins. If pre-lab on. is not complete at the beginning of lab, the student will be unable to participate in the lab sessi Questions: 1. (2pts) The first step of this experiment is to isolate chloroplasts from spinach. Review your Hill Reaction lab procedure. Briefly explain (2-3 sentences) how you will isolate chloroplasts. 2. (2pts) The second step of this experiment is to run SDS PAGE of your chloroplast extracts. Review your SDS PAGE lab procedure. Briefly explain (2-3 sentences) how SDS PAGE will separate proteins based on size. 3.(2pts) The final step of this experiment is to do a Western Blot. Explain (2-3 sentences) what happens during a Western Blot procedure. 4.(2pts) What is Alkaline Phosphatase and how is it used in a Western Blot procedure? (2pts) What will a Western blot tell us about a protein? How is this different from what SDS PAGE will tell us about a protein?

Explanation / Answer

1.The separation of chloroplasts can be done using a centrifuge. The spinach leaves are grounded in a buffer and later arranged in a centrifuge, the initial pellets are discarded off. The remaining supernatant is spun at a greater rpm, the pellets of this round will include the chloroplasts and are resuspended in the buffer solution.

2.   SDS PAGE divides proteins on the basis of their size, nevertheless proteins are supposed to be denatured firstly which allows them to be linear and also give them a negative charge. The proteins are next loaded in a gel with an electrical current. The proteins move within the gel leaving behind notable bands in which the larger proteins will not travel as far as the smaller proteins. The gels are later stained so that the bands become apparent and can be measured and compared to a ladder. the ladder was also loaded and the molecular weight of the protein can be calculated later.

3. for identifying proteins Western Blotting is beneficial which is dependent on the way they interact with antibodies. The proteins are conveyed onto a nitrocellulose membrane applying an electric current. It is later incubated with primary antibody of interest which is RuBisCo and following  RuBisCo it is later incubated with a secondary antibody that adheres to the primary. the secondary antibody is associated with an enzyme termed as Alkaline Phosphatase

4. Alkaline Phosphatase is an enzyme which is used in the Western Blot method to envision the band created from the primary antibody. It develops colour when it reacts with its substrate producing bands wherever the RuBisCo antibody ran on the SDS Page

5. to identify a protein by the communication of a protein with a particularly distinct antibody Western Blot helps us, however, the SDS PAGE helps us recognize proteins by their size.

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