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Characteristics and precautions of total protein extraction kit
NegotiableUpdate on 05/06
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Overview
The formula contained in the total protein extraction kit can effectively dissolve cell membrane components, including the cytoplasmic membrane, nuclear membrane, and various organelle membranes. The protease inhibitor mixture contained in this kit prevents the degradation of proteins by proteases, providing a guarantee for extracting high-purity proteins.
Product Details
Storage temperature2-8℃
Total protein extraction kitPrecautions
Protease inhibitors can also be stored at 2-8 ℃ before use without opening the lid. Store at -20 ℃ after opening and using.
2. Protease inhibitors are in a solid state at low temperatures of 2-8 ℃. After being removed from the refrigerator, they are returned to room temperature or briefly immersed in a water bath at 37 ℃. Once they become liquid, they are centrifuged to the bottom of the tube before opening the lid.
3. Please use up the reagents as soon as possible after unpacking!
2. Protease inhibitors are in a solid state at low temperatures of 2-8 ℃. After being removed from the refrigerator, they are returned to room temperature or briefly immersed in a water bath at 37 ℃. Once they become liquid, they are centrifuged to the bottom of the tube before opening the lid.
3. Please use up the reagents as soon as possible after unpacking!
Validity period
a year
detection method
WB,IP,co-IP,ELISA,EMSA 等
Applicable samples
Animal cells/tissues
Total protein extraction kitProduct Features
1. Easy to use, protein extraction from cells and tissues does not require pre-treatment such as grinding, repeated freeze-thaw cycles, ultrasonic fragmentation, etc.
2. The extraction process is simple and convenient, reducing the protein extraction time to 30 minutes to 1 hour.
3. Containing protein stabilizer, the extracted protein is stable.
When detecting protein concentration using UV, background interference is low.
5. The total protein extraction solution contains multiple effective ingredients, which can fully release cytoplasmic proteins, nuclear proteins, and membrane proteins, and can also bind to the released proteins to prevent precipitation.
6. Protease inhibitors inhibit protein degradation and optimize the formulation of protease inhibitors. The protease inhibitor mixture contains six independent protease inhibitors, AEBSF, Aprotinin, Leupeptin, Pepstatin A, Bestatin, and E-64, each of which can specifically inhibit one or several protease activities. The optimized composition of this mixture enables it to inhibit almost all important protease activities, including proteases, hemiproteases, aspartic proteases, alanyl aminopeptidases, etc.
2. The extraction process is simple and convenient, reducing the protein extraction time to 30 minutes to 1 hour.
3. Containing protein stabilizer, the extracted protein is stable.
When detecting protein concentration using UV, background interference is low.
5. The total protein extraction solution contains multiple effective ingredients, which can fully release cytoplasmic proteins, nuclear proteins, and membrane proteins, and can also bind to the released proteins to prevent precipitation.
6. Protease inhibitors inhibit protein degradation and optimize the formulation of protease inhibitors. The protease inhibitor mixture contains six independent protease inhibitors, AEBSF, Aprotinin, Leupeptin, Pepstatin A, Bestatin, and E-64, each of which can specifically inhibit one or several protease activities. The optimized composition of this mixture enables it to inhibit almost all important protease activities, including proteases, hemiproteases, aspartic proteases, alanyl aminopeptidases, etc.
Product Application
WB,IP,co-IP,ELISA,EMSA 等
Instrument preparation
1. Centrifuge
2. Oscillators
3. Homogenizer/homogenizer
4. Vortex mixer
5. Pipette
6. Refrigerator
7. Ice box
2. Oscillators
3. Homogenizer/homogenizer
4. Vortex mixer
5. Pipette
6. Refrigerator
7. Ice box
Reagent preparation
1. PBS buffer solution
2. Protein quantification kit
2. Protein quantification kit
Preparation of consumables
1. Centrifugal tube
2. Suction head
3. Disposable gloves
2. Suction head
3. Disposable gloves
Precautions for use
Before opening the cap, please briefly centrifuge the reagents in the rotary cap centrifuge tube, and shake the liquid on the inner wall of the cap to the bottom of the tube to avoid liquid spillage when opening the cap.
Protease inhibitors are in a solid state at 2-8 ℃. After being taken out of the refrigerator, they are returned to room temperature or briefly immersed in a water bath at 37 ℃. Once they become liquid, they are centrifuged to the bottom of the tube before opening the lid.
All reagents used during the experiment must be pre cooled; All appliances must be pre cooled in a -20 ℃ refrigerator. The entire process must keep the sample at a low temperature.
If precipitation occurs during the storage of protease inhibitors, it does not affect their use and can be used normally after dissolution.
If the reagent kit cannot be used up in a short period of time, the protease inhibitor mixture cannot be added to the extraction solution all at once.
Other protease inhibitor monomers can be added according to one's own experimental needs.
If downstream experiments are conducted to detect the enzyme activity of specific proteases or phosphatases, the extraction solution can be free of protease inhibitors or phosphatase inhibitors. It is important to maintain low-temperature operation during the extraction process and shorten the centrifugation time.
Beta actin, GAPDH, and Tubulin can be used as reference materials for Western Blot experiments.
The centrifuge speed can be expressed in two ways: relative centrifugal force (RCF, x g) and revolutions per minute (RPM, r/min). Some centrifuges have RPM and x g display switches, but some centrifuges do not have automatic switching function. The following formula is needed for conversion: g=r × 1.118 × 10-5 × rpm2 (r is the effective centrifugal radius, which is the length from the centrifuge axis to the center of the centrifuge collection tube bottom in centimeters). For example, if the speed is 3000rpm and the effective centrifugal radius is 10cm, the relative centrifugal force (RCF, × g) is=10 × 1.118 × 10-5 × 30002=1006.2 (× g).
Protease inhibitors are in a solid state at 2-8 ℃. After being taken out of the refrigerator, they are returned to room temperature or briefly immersed in a water bath at 37 ℃. Once they become liquid, they are centrifuged to the bottom of the tube before opening the lid.
All reagents used during the experiment must be pre cooled; All appliances must be pre cooled in a -20 ℃ refrigerator. The entire process must keep the sample at a low temperature.
If precipitation occurs during the storage of protease inhibitors, it does not affect their use and can be used normally after dissolution.
If the reagent kit cannot be used up in a short period of time, the protease inhibitor mixture cannot be added to the extraction solution all at once.
Other protease inhibitor monomers can be added according to one's own experimental needs.
If downstream experiments are conducted to detect the enzyme activity of specific proteases or phosphatases, the extraction solution can be free of protease inhibitors or phosphatase inhibitors. It is important to maintain low-temperature operation during the extraction process and shorten the centrifugation time.
Beta actin, GAPDH, and Tubulin can be used as reference materials for Western Blot experiments.
The centrifuge speed can be expressed in two ways: relative centrifugal force (RCF, x g) and revolutions per minute (RPM, r/min). Some centrifuges have RPM and x g display switches, but some centrifuges do not have automatic switching function. The following formula is needed for conversion: g=r × 1.118 × 10-5 × rpm2 (r is the effective centrifugal radius, which is the length from the centrifuge axis to the center of the centrifuge collection tube bottom in centimeters). For example, if the speed is 3000rpm and the effective centrifugal radius is 10cm, the relative centrifugal force (RCF, × g) is=10 × 1.118 × 10-5 × 30002=1006.2 (× g).
Usage
Total protein extraction from cell samples:
Suspension cell protein extraction:
1. Preparation of extraction solution:
Add 2 μ l of protease inhibitor mixture and 2 μ l of phosphatase inhibitor mixture to every 500 μ l of cold total protein extract, mix well, and set aside on ice.
2. Take 5 × 106 cells, centrifuge at 4 ℃ and 2500 × g for 5 minutes, carefully absorb the culture medium, and collect the cells as dry as possible.
3. Wash the cells twice with cold PBS and try to absorb the supernatant as much as possible after each wash.
4. Add 500 μ l of cold total protein extract to every 5 × 106-1 × 107 cells (approximately 50 mg cells/50 μ l cell precipitation volume), blow and mix well, shake at 4 ℃ for 20-30 minutes until the cells are fully lysed and there is no obvious cell precipitation.
Centrifuge at 4 ℃ and 12000 × g for 15 minutes.
6. Inhale the supernatant into another pre cooled clean centrifuge tube to obtain the total protein.
7. Quantify the protein extract and package it in a -80 ℃ refrigerator for storage or use directly for downstream experiments.
Protein extraction from adherent cells:
1. Preparation of extraction solution:
Add 2 μ l of protease inhibitor mixture and 2 μ l of phosphatase inhibitor mixture to every 500 μ l of cold total protein extract, mix well, and set aside on ice.
2. Carefully absorb the culture medium of adherent cells.
3. Wash the cells twice with cold PBS and try to absorb the PBS as much as possible after each wash.
4. Add an appropriate amount of cold total protein extract, shake for 15-30 minutes until the cells are fully lysed, scrape with a cell scraper, and draw the lysate into another clean centrifuge tube.
Centrifuge at 4 ℃ and 12000 × g for 15 minutes.
6. Inhale the supernatant into another pre cooled clean centrifuge tube to obtain the total protein.
7. Quantify the protein extract and package it in a -80 ℃ refrigerator for storage or use directly for downstream experiments.
Total protein extraction from organizational samples:
1. Preparation of extraction solution:
Add 2 μ l of protease inhibitor mixture and 2 μ l of phosphatase inhibitor mixture to every 500 μ l of cold total protein extract, mix well, and set aside on ice.
2. Take 50-100 mg of tissue sample, wash it with PBS, then cut it as much as possible with a knife, add 500 μ l of total protein extraction solution, and homogenize it with a tissue homogenizer/homogenizer until there are no visible solids.
3. Inhale the tissue homogenate into a pre cooled clean centrifuge tube and shake at 4 ℃ for 10-20 minutes.
4. Centrifuge at 4 ℃ and 10000-14000 × g for 15 minutes.
5. Inhale the supernatant into another pre cooled clean centrifuge tube to obtain the total protein.
6. Quantify the protein extract and package it in a -80 ℃ refrigerator for storage or use directly for downstream experiments.
Suspension cell protein extraction:
1. Preparation of extraction solution:
Add 2 μ l of protease inhibitor mixture and 2 μ l of phosphatase inhibitor mixture to every 500 μ l of cold total protein extract, mix well, and set aside on ice.
2. Take 5 × 106 cells, centrifuge at 4 ℃ and 2500 × g for 5 minutes, carefully absorb the culture medium, and collect the cells as dry as possible.
3. Wash the cells twice with cold PBS and try to absorb the supernatant as much as possible after each wash.
4. Add 500 μ l of cold total protein extract to every 5 × 106-1 × 107 cells (approximately 50 mg cells/50 μ l cell precipitation volume), blow and mix well, shake at 4 ℃ for 20-30 minutes until the cells are fully lysed and there is no obvious cell precipitation.
Centrifuge at 4 ℃ and 12000 × g for 15 minutes.
6. Inhale the supernatant into another pre cooled clean centrifuge tube to obtain the total protein.
7. Quantify the protein extract and package it in a -80 ℃ refrigerator for storage or use directly for downstream experiments.
Protein extraction from adherent cells:
1. Preparation of extraction solution:
Add 2 μ l of protease inhibitor mixture and 2 μ l of phosphatase inhibitor mixture to every 500 μ l of cold total protein extract, mix well, and set aside on ice.
2. Carefully absorb the culture medium of adherent cells.
3. Wash the cells twice with cold PBS and try to absorb the PBS as much as possible after each wash.
4. Add an appropriate amount of cold total protein extract, shake for 15-30 minutes until the cells are fully lysed, scrape with a cell scraper, and draw the lysate into another clean centrifuge tube.
Centrifuge at 4 ℃ and 12000 × g for 15 minutes.
6. Inhale the supernatant into another pre cooled clean centrifuge tube to obtain the total protein.
7. Quantify the protein extract and package it in a -80 ℃ refrigerator for storage or use directly for downstream experiments.
Total protein extraction from organizational samples:
1. Preparation of extraction solution:
Add 2 μ l of protease inhibitor mixture and 2 μ l of phosphatase inhibitor mixture to every 500 μ l of cold total protein extract, mix well, and set aside on ice.
2. Take 50-100 mg of tissue sample, wash it with PBS, then cut it as much as possible with a knife, add 500 μ l of total protein extraction solution, and homogenize it with a tissue homogenizer/homogenizer until there are no visible solids.
3. Inhale the tissue homogenate into a pre cooled clean centrifuge tube and shake at 4 ℃ for 10-20 minutes.
4. Centrifuge at 4 ℃ and 10000-14000 × g for 15 minutes.
5. Inhale the supernatant into another pre cooled clean centrifuge tube to obtain the total protein.
6. Quantify the protein extract and package it in a -80 ℃ refrigerator for storage or use directly for downstream experiments.
Common problem analysis
1. Low protein concentration?
When processing some tissue samples, there may be no lysis, resulting in low protein concentration. As long as the processing time of reagent A is appropriately extended. Under continuous oscillation conditions, even without an oscillator, it is possible to use a suction head to blow and mix evenly at intervals of a few minutes.
2. Slow cell lysis rate?
In order to fully ensure the activity of the extracted protein, the protective protein formula used in the extraction solution has mild lysis ability and a wide range of downstream applications. Extending the cracking time appropriately is sufficient.
3. What method is used to quantify protein?
Suggest using BCA method. It is not suitable to use the Bradford method because reagent A contains components that interfere with the Bradford method, resulting in inaccurate quantification. If dialysis treatment has already been carried out or the buffer system has been replaced with a desalination column, the Bradford method can be used for quantification.
4. Is there a gel like precipitate during extraction?
Sometimes a small amount of transparent gel like substance may appear in the processed product of protein extraction solution, which is a normal phenomenon. The transparent gel like substance is a complex containing genomic DNA and other components. Without detecting specific proteins that bind closely to genomic DNA, the supernatant can be directly centrifuged for subsequent experiments; If it is necessary to detect proteins that are particularly closely bound to the genome, ultrasound treatment can be performed at 300w/10 second intervals of 10 seconds for 3 minutes, followed by centrifugation to obtain the supernatant for subsequent experiments. When detecting some common transcription factors, such as NF kappaB, p53, etc., ultrasound treatment is not necessary.
5. Does the extracted protein have activity?
This reagent kit does not contain ionic detergent components, does not damage the structure of proteins, and does not disrupt the original interactions between proteins. Proteins maintain their natural conformation and activity.
When processing some tissue samples, there may be no lysis, resulting in low protein concentration. As long as the processing time of reagent A is appropriately extended. Under continuous oscillation conditions, even without an oscillator, it is possible to use a suction head to blow and mix evenly at intervals of a few minutes.
2. Slow cell lysis rate?
In order to fully ensure the activity of the extracted protein, the protective protein formula used in the extraction solution has mild lysis ability and a wide range of downstream applications. Extending the cracking time appropriately is sufficient.
3. What method is used to quantify protein?
Suggest using BCA method. It is not suitable to use the Bradford method because reagent A contains components that interfere with the Bradford method, resulting in inaccurate quantification. If dialysis treatment has already been carried out or the buffer system has been replaced with a desalination column, the Bradford method can be used for quantification.
4. Is there a gel like precipitate during extraction?
Sometimes a small amount of transparent gel like substance may appear in the processed product of protein extraction solution, which is a normal phenomenon. The transparent gel like substance is a complex containing genomic DNA and other components. Without detecting specific proteins that bind closely to genomic DNA, the supernatant can be directly centrifuged for subsequent experiments; If it is necessary to detect proteins that are particularly closely bound to the genome, ultrasound treatment can be performed at 300w/10 second intervals of 10 seconds for 3 minutes, followed by centrifugation to obtain the supernatant for subsequent experiments. When detecting some common transcription factors, such as NF kappaB, p53, etc., ultrasound treatment is not necessary.
5. Does the extracted protein have activity?
This reagent kit does not contain ionic detergent components, does not damage the structure of proteins, and does not disrupt the original interactions between proteins. Proteins maintain their natural conformation and activity.
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