Plant Tissue Membrane Vesicle (RSOV and IOV) Preparation Kit Product Manual (Chinese version) The main purpose Plant tissue membranous vesicle (RSOV and IOV) preparation reagent is a kind of normal outward ventral vesicle (RSOV) and medial valgus membrane designed to separate from plant tissues by differential centrifugation. An authoritative and classic technical approach to the composition of In-side out (IOV). The technology has been carefully developed and successfully tested. It is suitable for the preparation of various plant tissues, including membranous vesicles such as seeds, leaves, roots, cotyledons, epicotyls, and the like. The preparation has high yield and can be used for membrane channel, signal transduction mechanism, membrane protein distribution and the like. The product does not contain contaminating proteases, that is, ready to use, stable performance, excellent separation yield and purity.
technical background Membrane vesicles are divided into normal ventral vesicles (Right-side out; RSOV) and medial valvular vesicles (In-side out; IOV). Normal outward membranous vesicles remove the cytoplasmic contents and lose their fine internal metabolic system, while maintaining the original phase of the plasma membrane, which helps to provide a suitable substrate without degradation and metabolism, establishing a dynamic mechanism. Transport or uptake studies are performed, such as microsomal fractions for V-type ATPase studies and mitochondrial components for F-type ATPase studies. The medial epidural vesicle is a vesicle-like structure that circumscribes the inner membrane and valgus after lysis of cells and organelles. It is widely used in the study of primary transport systems.
product content Reagent A 100 ml balance solution (Reagent B) 60 ml preservation solution (Reagent C) 20 ml product manual 1 part
storage method Preservation solution (Reagent C) in the refrigerator at -20 ° C, the rest is stored in the refrigerator at 4 ° C; effective guarantee for June
User-supplied 15 ml conical centrifuge tube: container for sample handling
1.5 ml centrifuge tube: container for sample handling and storage
4°C ultracentrifuge: for separation of membranous vesicles
DOUNCE homogenizer: used to lyse tissue
Experimental procedure I.
Normal outward membranous vesicle separation (mitochondria and microsomes) - Prepare fresh plant tissue (leaves, grains, seeds, etc.) and weigh to determine 1 gram of tissue weight
- (selection step) into a pre-cooled 15 ml conical tube
- (Selection step) Add 3 ml of cleaning solution ( Reagent A ) to wash once
- Move into a liquid nitrogen cryotube
- Immediately put in a liquid nitrogen tank overnight
- Removed from the liquid nitrogen tank the next day and immediately crushed the tissue into a powder (fastest): Do not freeze or thaw the tissue .
- Put in a 15 ml conical tube
- Add 3 ml of pre-cooled balance solution (Reagent B)
- Vortex for 5 seconds, mix thoroughly
- Immediately placed in a pre-cooled DOUNCE homogenizer
- Homogenize the tissue with a grinding rod in the ice trough (about 80 times)
- Transfer all tissue homogenates into a 15 ml conical tube
- Place in a 4°C benchtop centrifuge for 10 minutes at 1000g
- Carefully remove the supernatant to another new pre-cooled 15 ml conical tube
- Place in a 4°C benchtop centrifuge for 10 minutes at a speed of 10,000g
- Carefully remove the supernatant into 2 new 1.5 ml centrifuge tubes while preserving the pellet
- Add 500 μl of Reservation Solution (Reagent C) to the pellet tube - this is the mitochondrial RSOV component (Note: Can be used for F-type ATPase studies)
- The two 1.5 ml centrifuge tubes containing the supernatant were placed in a 4 ° C ultracentrifuge for 60 minutes at a speed of 100,000 g.
- Carefully remove the supernatant
- Add 500 μl of Reservation Solution (Reagent C) - this is the microsomal RSOV component (Note: Can be used for V-type ATPase studies)
- Pipette 10 μl for protein quantitation (Note: Bradford Protein Concentration Quantitation Kit -30030.1 is recommended)
- Immediately put in a -70 ° C refrigerator
Second, the medial valgus vesicle separation (plasma membrane separation) - Prepare fresh plant tissue (leaves, grains, seeds, etc.) and weigh to determine 1 gram of tissue weight
- (selection step) into a pre-cooled 15 ml conical tube
- (Selection step) Add 3 ml of cleaning solution ( Reagent A ) to wash once
- Move into a liquid nitrogen cryotube
- Immediately put in a liquid nitrogen tank overnight
- Removed from the liquid nitrogen tank the next day and immediately crushed the tissue into a powder (fastest): Do not freeze or thaw the tissue .
- Put in a 15 ml conical tube
- Add 3 ml of pre-cooled balance solution (Reagent B)
- Vortex for 5 seconds, mix thoroughly
- Immediately placed in a pre-cooled DOUNCE homogenizer
- Homogenize the tissue with a grinding rod in the ice trough (about 80 times)
- Transfer all tissue homogenates into a 15 ml conical tube
- Place in a 4°C benchtop centrifuge for 10 minutes at 1000g
- Carefully remove the supernatant to another new pre-cooled 15 ml conical tube
- Place in a 4°C tabletop centrifuge for 30 minutes at a speed of 10,000g
- Carefully remove the supernatant
- Add 500 μl of Reservation Solution (Reagent C) - this is the plasma membrane RSOV component (Note: Can be used for P-type ATPase studies)
- Freeze-thaw 4 times in continuous -70 ° C to 37 ° C - this is the plasma membrane IOV component
- Pipette 10 μl for protein quantitation (Note: Bradford Protein Concentration Quantitation Kit -30030.1 is recommended)
- Immediately put in a -70 ° C refrigerator
- (Selection step) Perform IOV component purification (Recommended IOV Membrane Vesicle High Purity Kit -10170)
Precautions - This product is operated 20 times (1 gram plant tissue)
- All operations must be performed at 4 ° C or below
- It is recommended to use a sufficient amount of sample
- Usually 1 gram of plant tissue has a microsomal component content of 50 mg protein, a mitochondrial component content of 5 mg protein, and a 100 mg plasma membrane.
5. The plasma membrane IOV component is a crude extraction product containing RSOV and unclosed plasma membrane. If more than 95% high purity IOV is required, IOV membrane vesicle high quality purification kit -10170 is recommended.
6. Purified samples avoid repeated freezing and thawing
- The company provides a series of ion transport channel transport function (transport / uptake) analysis reagent products
Quality Standard - This product has been certified to be stable.
- This product has been identified to separate components to maintain normal activity
- This product has been identified as free of contaminating proteases and ribozymes.
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