Purification of Green Fluorescent Protein Introduction:
Western Blot analysis was performed as described in the Materials and Methods. A band appeared in the lanes containing normal cells and in two of the tumors that matches the expected molecular weight of P53, whereas no band appeared in the other two lanes containing tumor cells.
These data indicated that P53 protein is missing from half of the tumors analyzed here. Even though P53 is detected in two of the tumors, it is possible that P53 is not active in these tumors. Our assay only determines if P53 protein is missing.
Just because P53 is present does not mean that it is active. Thus, the two tumors that contained P53 may contain inactive protein. To determine if the P53 in the two tumors is active, we would have to isolate the P53 protein and test it for its ability to halt cells in G1 or to induce apoptosis of the cells.
Some tumors never lose P53 activity and thus, must become tumors through another pathway that doesn't include P It would be interesting to look for the loss of other tumor suppressor proteins from these tumors. The P53 protein is involved in cell cycle regulation and apoptosis. When the damage is repaired, P53 levels decrease and the cell can proceed into S phase.
If the damage is too great to be repaired P53 can initiate apoptosis programmed cell death. First, the gene encoding P53 can be mutated or lost. Second, the p53 gene can be silenced or not expressed. Third, the protein is expressed but its function is blocked.
Fourth, the protein is expressed but is quickly degraded before it can perform its action. We performed Western Blot Analysis to determine if the protein was present in the cells. Antibodies directed against the protein of interest are used to detect the protein.
Our results lend support to the idea that P53 acts as a tumor suppressor protein. Isolation of Proteins from Cells. The resulting pellet was resuspended in 1 ml of distilled water and dialyzed against distilled water overnight.
The other part of the gel, containing the protein samples from the two cell lines was loaded onto an electroblotting apparatus. The gel was blotted onto Nylon at 90 V for 2 hours. Then the membrane was washed 3 times 5 - 10 minutes each time in wash solution 1 X TBS and 0.
The blot was then washed 3 times minutes each with wash solution. Goat anti-rabbit antibodies conjugated to horseradish peroxidase enzyme were added to the antibody incubation buffer at room temperature for one hour. The blot was washed 3 times minutes each time in wash buffer.
The ECL chemiluminenscence kit was used for detection of antibodies.
Solutions 1 and 2 were mixed and poured on blot for 1 minute. This resulting solution was carefully poured off and the blot was exposed to autoradiography film. They ranged from 1.The day before lab, pick 4 transformants from each ligation with separate sterile toothpicks, and inoculate separate culture tubes containing liquid medium (LB+Kan).
Grow overnight with shaking at 37ºC. ©UF Column Chromatography • A separation method in which different components of a mixture travel through the resin of a column differently. The ChemiDoc XRS+ system is based on CCD high-resolution, high-sensitivity detection technology and modular options to accommodate a wide range of samples and support multiple detection methods including fluorescence, colorimetry, densitometry, chemiluminescence, and chemifluorescence.
In the GFP MS/MS experiments, what are the charge states of the product ions?
Are they For your lab report, please turn in answers to each of the lab questions, including all relevant graphs, calculations and discussions. Title: Microsoft Word - barnweddingvt.com Author.
Using PCR to detect a single gene (GFP): In this lab investigation students will learn about a technique called polymerase chain reaction (PCR) that allows us to examine a very small piece of DNA.
The piece of DNA that is replicated is called the Green Fluorescent Protein (GFP) gene. Lab exercises with GFP include: high speed centrifuging to concentrate the cells, cell lysis, ammonium sulfate precipitation to collect the protein fraction, or, alternatively, three-phase partitioning (a seldom used, but spectacular method for purifying proteins).