Plant tissue extraction kit. For extraction of soluble protein and other biomolecules and metabolites from plant tissues.

Transcription

1 ab Plant tissue extraction kit Instructions for use: For extraction of soluble protein and other biomolecules and metabolites from plant tissues. This product is for research use only and is not intended for diagnostic use. Version 3 Last Updated 16 December 2015

2 Table of Contents INTRODUCTION 1 1. BACKGROUND 1 2. ASSAY SUMMARY 2 GENERAL INFORMATION 3 3. PRECAUTIONS 3 4. STORAGE AND STABILITY 3 5. LIMITATIONS 3 6. MATERIALS SUPPLIED 4 7. MATERIALS REQUIRED, NOT SUPPLIED 4 8. TECHNICAL HINTS 4 ASSAY PREPARATION 5 9. REAGENT PREPARATION SAMPLE PREPARATION 5 ASSAY PROCEDURE ASSAY PROCEDURE 6 DATA ANALYSIS TYPICAL DATA 7 RESOURCES NOTES 9

3 INTRODUCTION INTRODUCTION 1. BACKGROUND Abcam s Plant tissue extraction kit (ab206999) enables effective extraction of soluble protein and metabolites from a range of dry and fresh plant tissues without the use of liquid nitrogen. The extracted proteins and metabolites can be used in applications such as western blotting, 2-D gel electrophoresis, immunoprecipitation assay, enzymatic activity assays, quantitation of metabolites etc. The kit includes optimized Extraction Buffer to extract plant protein and metabolites, Protease Inhibitor Cocktail to prevent protein degradation and Homogenization Pestles and Tubes to homogenize the plant tissue. This easy-to-use and rapid procedure ensures consistent protein yield (~ 2-9 mg/ml, depending on the sample type). ab Plant tissue extraction kit 1

4 INTRODUCTION 2. ASSAY SUMMARY Extraction Protein Quantitation ab Plant tissue extraction kit 2

5 GENERAL INFORMATION GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at -20 C in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in sections 6 and LIMITATIONS Kit intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. ab Plant tissue extraction kit 3

6 GENERAL INFORMATION 6. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) Plant Extraction Buffer 25 ml -20 C -20 C Protease Inhibitor Cocktail 1 ml -20 C -20 C (200X) Homogenization Pestles 3-20 C or RT -20 C or RT Homogenization tubes 3 X 1.5 ml -20 C or RT -20 C or RT 7. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: Blades and tweezers Microcentrifuge BCA Protein Assay Kit (e.g., ab102536) 8. TECHNICAL HINTS This kit is sold based on number of tests. A test simply refers to a single extraction experiment. The starting amount of tissue or cells for a single experiment will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Homogenization Pestles and Tubes are reusable. After each use, rinse 3 times with dh 2 O and air-dry. ab Plant tissue extraction kit 4

7 ASSAY PREPARATION ASSAY PREPARATION 9. REAGENT PREPARATION 9.1. Plant extraction buffer: Ready to use as supplied Protease Inhibitor Cocktail: Ready to use as supplied Homogenization Pestles: Homogenization Pestles can be stored at room temperature or - 20 C Homogenization Tubes: Homogenization Pestles can be stored at room temperature or - 20 C. 10.SAMPLE PREPARATION Thaw frozen plant tissue on ice before cutting into small pieces To soften dry plant tissue such as seeds, roots etc., completely soak the tissue in dh 2 O and incubate at 4 C for 1-2 days. ab Plant tissue extraction kit 5

8 ASSAY PROCEDURE ASSAY PROCEDURE 11.ASSAY PROCEDURE Extraction Add 5 µl of Protease Inhibitor Cocktail to 995 µl of cold Plant Extraction Buffer before use and keep on ice to chill Clean the tissue with dh 2 O and remove excess water Weigh the fresh or frozen tissue (~ mg) and cut into small pieces with blade Transfer the tissue pieces into the 1.5 ml Homogenization Tube provided and add ~ μl ice cold Plant Extraction Buffer with Protease Inhibitor Cocktail Homogenize the tissue on ice by rotating the pestle in the 1.5 ml Homogenization Tube 60 times Keep the Homogenization Tube on ice for 10 minute Centrifuge at 10,000 x g, 4 C for 5 minutes and collect the supernatant Protein Quantitation Measure the protein concentration using a BCA Protein Assay Kit Keep the plant tissue homogenate on ice for immediate use, or store at -80 C for future use. ab Plant tissue extraction kit 6

9 DATA ANALYSIS DATA ANALYSIS 12. TYPICAL DATA Figure 1: SDS-PAGE of plant tissue lysates prepared using ab206999: 25 μl of lysate from lettuce (20 μg, lane 2), green pepper (20 μg, lane 3), avocado (40 μg, lane 4) and orchid leaf (10 μg, lane 5) were loaded onto a 4-20% SDS- PAGE and, following electrophoresis, stained with Coomassie blue. Lane 1: Marker. Figure 2: Measurement of Glucose-6-Phosphate in orchid leaf lysate (5 μg) using a G6P Fluorometric Assay Kit. The lysate was prepared using ab ab Plant tissue extraction kit 7

10 DATA ANALYSIS Figure 3: Measurement of glucose-6-phosphate dehydrogenase activity in orchid leaf lysate (0.6 μg) using a G6PDH Activity Assay Kit. The lysate was prepared using ab ab Plant tissue extraction kit 8

11 RESOURCES RESOURCES 13.NOTES ab Plant tissue extraction kit 9

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