大肠杆菌蛋白表达试剂盒(阿拉伯糖诱导)

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表达试剂盒产品基本信息

产品名称: 大肠杆菌蛋白表达试剂盒(阿拉伯糖诱导)
英文名称:
L-arabinose induced E.coli expression kit
 
产品目录号: K300-01
内容与规格:
6X30g 表达专用培养基粉末(每袋可配成1L液体培养基)
5x1mL SOC(感受态细胞转化)
4X6 mL  L-阿拉伯糖梯度溶液(无菌)
 
保存条件: 室温




产品描述:

阿拉伯糖诱导E.coli表达试剂盒试用于pBAD系列载体、pDEST15、pDEST17、
pDEST24等阿拉伯糖诱导的大肠杆菌表达载体。

 
Biofeng Lab提供的试剂盒内含有SOB培养基,营养密度高,是高效的表达用
培养基。4个浓度梯度的L-阿拉伯糖溶液,无需稀释,有助于快速建立最佳
表达方案,提高表达水平。
 


阿拉伯糖诱导的蛋白表达实验流程

小试实验
 
In addition to testing your transformants, we recommend
that you include cells without a vector as a negative control.
 
1. For each transformant or control, inoculate 2 ml of SOB or
LB medium containing 50–100 μg/ml ampicillin with a
single recombinant E. coli colony.

2. Grow cells overnight on a 37°C shaking incubator at
225–250 rpm. The cells should have an optical density at
600 (OD600) of 1~2.
 
3. Label five tubes 1 through 5 and add 10 ml of SOB or LB
medium containing 50–100 μg/ml ampicillin.
 
4. Inoculate each tube with 0.1 ml of the overnight culture.
 
5. Grow the cultures in the tubes on a 37°C shaking
incubator (use vigorous shaking) Grow cells to an
OD600 of ~0.5 (the cells should be in mid-log phase).
 
6. While the cells are growing, prepare four 10-fold serial
dilutions of 20% L-arabinose with sterile water using
aseptic technique (e.g. 2%, 0.2%, 0.02%, and 0.002%).
 
7. Take a 1 ml aliquot of grown cells from each tube, and
place each aliquot in separate microcentrifuge tubes.
Centrifuge at maximum speed in a microcentrifuge for
30 seconds, and aspirate the supernatant.
 
8. Freeze the cell pellet at –20°C. This is the zero time pointsample.
 
9. Using the solutions prepared in Step 6, add the following
volumes of L-arabinose to the five 9 ml cultures.
 
Note: It is only necessary to test the highest concentration of L-arabinose.


Tube Stock Solution Volume(ml) Final Concentration
0.002% 0.9 0.00002%
0.02% 0.9 0.0002%
0.2% 0.9 0.002%
2% 0.9 0.02%
20% 0.9 0.2%










10. Grow at 37°C with shaking for 4 hours.
 
11. Take 1 ml samples at 4 hours and treat samples as in
Steps 7 and 8. You will have a total of 10 samples for each
transformant and two samples for the negative control.
 
12. Proceed to Analyzing Samples, next page.

分析小试结果
 
Before starting, prepare SDS-PAGE gels  to analyze all the
samples you collected.

 
Note: If you wish to analyze your samples for soluble
protein, see
the section below.
 
1. When all the samples have been collected from Steps 8
and 11 in the Pilot Expression, resuspend each pellet in
100 μl of 1X SDS-PAGE sample buffer.
 
2. Heat for 5 minutes at 70°C and centrifuge briefly.
 
3. Load 5–10 μl of each sample on an SDS-PAGE gel and
electrophorese. Save your samples by storing at –20°C.


判断目的蛋白的可溶性
 
1. Thaw and resuspend each pellet in 500 μl of Lysis Buffer.
 
2. Freeze sample in dry ice or liquid nitrogen and then thaw
at 42°C. Repeat 2 to 3 times.
Note: To facilitate lysis, you may need to add lysozyme or
sonicate the cells.
 
3. Centrifuge samples at maximum speed in a
microcentrifuge for 1 minute at +4°C to pellet insoluble
proteins. Transfer supernatant to a fresh tube and store
on ice.
 
4. Mix together equivalent amounts of supernatant and 2X
SDS-PAGE sample buffer and boil for 5 minutes.
 
5. Add 500 μl of 1X SDS-PAGE sample buffer to the pellets
from Step 3 and boil 5 minutes.
 
6. Load 10 μl of the supernatant sample and 5μl of the pellet
sample onto an SDS-PAGE gel and electrophorese.

确定最佳表达方案

To determine the success of your expression experiment,
you may want to perform the following types of analyses:
 
1. Stain the polyacrylamide gel with Coomassie blue and
look for a band of increasing intensity in the expected size
range for the recombinant protein. Use the uninduced
culture as a negative control.
 
2. Perform a Western blot to confirm that the overexpressed
band is your desired protein.
 
3. Determine the approximate arabinose concentration for
maximum expression.

检测目的蛋白
 
To detect expression of your recombinant fusion protein by
Western blot analysis, you may use antibodies against the
appropriate epitope  or an antibody to your protein of interest.
 
Once you have detected expression of your protein of
interest, you may wish to perform experiments to further
optimize expression. Use the Pilot Expression protocol
, but vary the L-arabinose concentration over a
smaller range. For example, if you obtained the best
expression at 0.002%, try 0.0004%, 0.0008%, 0.001%,
0.004%,
and 0.008%.
 
You may also perform a time course of induction to
determine if varying the time increases expression. Take
time points every hour, over a 5 to 6 hour period.
If your protein is insoluble, you may wish to analyze the
supernatant and pellet of lysed cells when you vary the
L-arabinose concentration .
 
Remember to store your cell lysates at –20°C.
 
毒性蛋白的表达
 
To ensure low levels of expression, you may want to utilize
glucose to further repress the araBAD promoter. Follow the
Pilot Expression protocol (page 9) using SOB or LB
containing 50–100 μg/ml ampicillin plus glucose at all steps
.
 

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