Questions: 1. (2pts) The first step of this experiment is to isolate chloroplast
ID: 64902 • Letter: Q
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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) L 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? 5. (2pts) will a Western blot tell us about a protein? How s different from what SDS PAGE will tell us about a protein?Explanation / Answer
Based on the given data,
1)
The plants produce carbohydrates (food) by consuming carbon dioxide, sunlight and water. This process is called photosynthesis and oxygen is released as a bi-product of photosynthesis.
According to the Hill reaction lab procedure, Robert Hill demonstrated that isolated chloroplasts can produce oxygen in the absence of carbon dioxide. He used artificial electron acceptor (dye) for demonstration of Hill reaction. First he isolated chloroplasts, then added the 2, 6-dichlorophenolindophenol, which has the ability to change color after reduction (blue to colorless). At 600 nm, the change absorbance explains the hill effect.
To isolate chloroplast, first we should take 30 grams of leaves, which then cut into small pieces. To these add 1X chloroplast isolation buffer. Then it is subjected to blending. After blending collect liquid and centrifuge it at 200 gravitons for 3 min. Then collect supernatant, again it centrifuge it at 1000 gravitons for 7 min. Now, take the pellet and resuspend it in 1X chloroplast isolation buffer. Now, one can see the layer of chloroplasts.
2)
SDS is an ionic detergent, it acts on proteins and breaks their ionic and hydrogen bonds this makes conformational change in proteins (tertiary or quaternary to primary) completely and provides the negative charge to protein. After providing negative charge all formed micelles trap the proteins.
The SDS PAGE is a gel electrophoresis method, it separate proteins based on molecular weight. The low molecular weight proteins go down and high molecular weight proteins are present at upper region.
3)
In Western blotting, the protein mixture is applied to a gel electrophoresis in a carrier matrix (SDS (sodium dodecyl sulfate)-PAGE) to separate the proteins on the basis of their size and charge. The separated protein bands are then transferred to a carrier membrane (e.g. nitrocellulose or nylon).
In Western blotting, one can use primary and secondary antibodies. The primary antibody used to recognize the target protein in a Western blot and sometimes this is not directly detectable. So, we add secondary antibodies to detect the target antigen (indirect detection). Basically, antibodies used because they are globular proteins that recognize specific proteins by identifying its amino acid sequence and with the help of this we can able to detect our desired protein.
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