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Team:SUSTC-Shenzhen/Notebook/A-B Toxin/Working Note
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date=2014| | date=2014| | ||
goal=--to purify the protein}} | goal=--to purify the protein}} | ||
| + | =8.24~8.30= | ||
| + | ==Results:== | ||
| + | According to the preview work procedure, we do the experiment. | ||
| + | ===8.28=== | ||
| + | In the SDS-PAGE gel elution stage, we use the new protocol | ||
| + | ====New elution protocol==== | ||
| + | 1.Use the Elution buffer(10% acetic acid, 40% absolute alcohol and 50% ddH2O) to do the elution(heating 5mins in microwave oven) | ||
| + | |||
| + | 2.Use the ddH2O to do the elution(heating 5mins in microwave oven) | ||
| + | ==Next week working plan== | ||
| + | 1.The quantitative analysis of protein, the refolding of purified proteins and search some useful documents. | ||
| + | |||
| + | 2.Increase the whole bacteria system to 800mL, and extract enough amount of protein in one week. | ||
| + | |||
=8.31~9.6= | =8.31~9.6= | ||
==Summary: == | ==Summary: == | ||
Revision as of 06:38, 12 October 2014
Notebook
Element of an endeavor
Contents |
A-B Toxin
2014 --to purify the protein8.24~8.30
Results:
According to the preview work procedure, we do the experiment.
8.28
In the SDS-PAGE gel elution stage, we use the new protocol
New elution protocol
1.Use the Elution buffer(10% acetic acid, 40% absolute alcohol and 50% ddH2O) to do the elution(heating 5mins in microwave oven)
2.Use the ddH2O to do the elution(heating 5mins in microwave oven)
Next week working plan
1.The quantitative analysis of protein, the refolding of purified proteins and search some useful documents.
2.Increase the whole bacteria system to 800mL, and extract enough amount of protein in one week.
8.31~9.6
Summary:
We did the protein refolding and a massive of protein purify.
Protein refolding:
9.2-9.3
tested the concentration of protein, and we used the Bradford.
9.3-19:00~21:00
added DTT (according to the protocol, make the mixture of DTT and denatured protein have a concentration of 3mM) to reduce the disulfide linkage in the objective protein.
9.3-21:00~9.5-21:00
added refolding buffer (the combination is do as the protocol says, but because of the low concentration of proteins, we used a proportion of 1mL protein solution：25mL refolding buffer ) The test of activity of protein: (we haven’t gotten the filter membrane yet, so it may be delayed)
Massive protein preparation:
8.31-9.4
We did as the improved protocol, but due to some mistake in the gradient of imidazole, the gel results look stranger compare to the results before. [two pictures]
The next-two-week-plan：
Maybe we should extract the protein again, and make sure our time-control and procedures are all right.
Also, we will cooperate with the plasmid constructing and cell line building group, so that we can test the activity of protein, and if the activity is reasonable, we can further use it in the cell line. If it did badly with gal4 sequences, we will do as the Prof. Wei advised, to use the filter membrane at the beginning.
And then, our protocol still has some problems, and we will talk about what we did to the prof.Zhang. He suggested that our purify process may be worry, so if the activity of our protein is not so good, we will do this experiment again.
Last but not least, we will start to formal the working plans and experiment notes before, and upload them to the wiki.
