For cells:
Note: Take an appropriate amount of lysate, pre-cool with 4 ° C, add enzyme inhibitors within a few minutes before use.
1. Adherent cells , cells were scraped, counted, and 5×106 cells were collected, centrifuged at 600g for 5 minutes, the supernatant was aspirated, the cells were resuspended in pre-cooled PBS, and the cells were collected by centrifugation at 600g for 5 minutes. After the supernatant was aspirated, 0.5 ml of the pre-cooled lysate was added, pipetted several times, incubated on ice for 30 minutes, centrifuged at 10,000 g for 10 minutes, and the supernatant was taken as a protein extract.
Note: The lysate can be directly added to the cultured cells to lyse the cells, as follows:
A. Carefully aspirate the culture solution with a pipette.
B. Add an appropriate amount of pre-cooled PBS,
C. Carefully aspirate the PBS with a pipette.
D. Add the lysate according to the table below and incubate on ice for 30 minutes.
E. The lysate was transferred to a centrifuge tube, centrifuged at 10,000 g for 10 minutes, and the supernatant was taken as a protein extract.
Board size / surface area | Reagent dosage |
100mm | 500~1000ul |
60mm | 250~500ul |
6-well plate | 200~400ul per well |
24-well plate | 100~200ul per well |
96-well plate | 50~100ul per well |
2. Suspend the cells , count, and collect 5 × 106 cells, centrifuge at 600g for 5 minutes, try to absorb the supernatant, resuspend the cells with pre-cooled PBS, centrifuge the cells for 5 minutes at 600g, collect the supernatant as much as possible, and add 0.5. The pre-cooled lysate of ml was pipetted several times, incubated on ice for 30 minutes, centrifuged at 10,000 g for 10 minutes, and the supernatant was taken as a protein extract.
For the organization:
Note: Take an appropriate amount of lysate, pre-cool with 4 ° C, add enzyme inhibitors within a few minutes before use.
1. The surgically removed tissue block is quickly placed in pre-cooled saline, rinsed several times, the tissue blood is washed, the tissue surface fluid is blotted with filter paper, and the tissue is cut into several smaller tissue blocks.
2. Weigh the tissue, according to the tissue net weight (g): lysate (ml) = 1:10, add the corresponding volume of lysate for homogenization, (if the lysis is not enough, you can add more lysate, if A high concentration of protein sample is required, and the amount of lysate can be appropriately reduced).
3. The homogenizer was homogenized, visually observed without obvious tissue blocks, and incubated on ice for 30 minutes.
4. Centrifuge at 10,000 g for 10 minutes and take the supernatant as a protein extract.
Example of results:
M N M N M N
DSG3 HSP90 PCNA
M:Hela Cells N:A549 Cells
As shown in the figure: Enhanced RIPA lysate extracts cell proteins, BCA determines protein concentration, loads 20 ug, electrophoresis, transfer membrane, incubates Bude's primary antibody, and uses Dr. De ECL luminescence to develop color.
Troubleshooting:
problem | the reason | Solution |
Low total protein | Some cells are more difficult to lyse | Ensure that the cell pellet is completely suspended in the enhanced RIPA buffer and incubated for a longer period of time, sonicated to increase yield |
Low protein concentration | More lysate | Reduce the amount of lysate |
Proteolysis | No protease inhibitor added | Add protease inhibitor |
Less phosphorylated protein | High phosphatase activity | Phosphatase inhibitor |
The performance of the Baby Monitor Security Camera
The performance of the baby monitor is reflected by its built-in parameters and function combination. The following shows some properties that a high-quality and applicable baby monitor should have. The requirements for baby monitors in this part apply to all audio/video, digital/analog home baby monitors and wireless baby monitors, and are not changed due to the combination mode of the monitoring end and the control end.
The size shall be as small as possible: the small baby monitor (monitoring end and control end) is more convenient to install and carry in the family.Low power display: the baby monitor, especially the monitoring terminal, must work continuously for a long time. Once the product is low in power, it cannot remind parents to charge it in time. When the power continues to be consumed until it is exhausted, the control terminal will be unable to continue to receive any signal.
Voice trigger prompt: Voice trigger means that once the monitoring terminal detects the noise caused by the abnormal behavior of infants or children, it will be displayed at the control terminal through the noise prompt lamp.
The transmission signal frequency can be adjusted: the monitoring end of the baby monitor provides audio and video digital signals to the control end by transmitting wireless signals. If the transmission frequency of the monitoring end is not selected properly, it will produce electromagnetic interference with other household appliances, digital equipment, etc., which will affect the normal use of the product. Therefore, it is necessary to select a baby monitor that allows users to adjust the frequency of the transmitted signal at the monitoring end.
Adjustable volume: a friendly baby monitor will provide a volume selection knob or button at the control end. When parents are in an environment with loud background sound, they can hear the activity sound of babies and children by turning up the volume; When it is late at night or in a quiet environment, the low volume will not affect the normal rest of the people around you when you are answering normally.
VOX function: VOX is the abbreviation of sound activation start. The monitor with VOX function is in standby mode under normal conditions, and the screen and horn stop working, thus reducing the energy consumption of the product; Once it is detected that the monitored object has an abnormal sound exceeding the set standard, it will immediately flash its alarm lamp at the control terminal or directly light up the screen, and simultaneously play the audio and video information sent by the monitoring terminal.
Digital signals and analog signals: baby monitors using digital signals, such as Baohushen baby monitors, will have better transmission efficiency and provide users with clearer sound and picture quality; Moreover, digital signals can be encrypted to prevent being intercepted and interpreted by other receivers, effectively protecting users' family privacy; The electromagnetic signal interference of digital signal will be lower than that of analog signal, so as not to affect the normal work of other household appliances and equipment.
Monitoring end extension: The baby monitor with a friendly user experience should allow users to use one control end to manage the activities of several monitoring ends at the same time, especially in some families with multiple children. This good scalability not only reduces the trouble of carrying multiple control ends at the same time, but also reduces the objective additional expenses for these families.
Temperature display: some products will have the function of realizing the current indoor temperature, and even can set a critical value. Once the room temperature exceeds or falls below the critical value, an alarm signal will be sent. This is a very considerate function for temperature sensitive newborn children or sick children.
Intercom function: The high-end baby monitor allows parents to have a direct conversation with the baby (child) at one end of the monitoring end through the control end, so that children who are alone can provide psychological comfort. If children have any temporary needs or words, they can be known without bothering the guardian to run to the child.
Signal range: theoretically, the signal sent by the monitoring terminal can be received by the remote control terminal, which is more convenient for users to use the baby monitor flexibly. Now more and more families live in large family rooms, even multi-storey villas, and the baby monitor with strong signal receiving ability provides these families with a more convenient way of use. Parents no longer need to set the room beside the children in order to see and hear their children's every move. They can even take the control terminal to go out for a short walk!
Power supply mode: The baby monitor with built-in battery can automatically switch to the battery power supply mode when the external AC power supply is suddenly stopped, so that the user will not be affected to continue to monitor the child in the power failure environment.
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Shenzhen Fuvision Electronics Co., Ltd. , https://www.szsolarcamera.com