ab211044 Lipid Extraction Kit (Chloroform-Free) Instructions for use: For chloroform-free lipid extraction from plasma, serum, cultured cells and tissues. View kit datasheet: www.abcam.com/ab211044 (use www.abcam.cn/ab211044 for China, or www.abcam.co.jp/ab211044 for Japan) This product is for research use only and is not intended for diagnostic use. Version 3 Last Updated 20 December 2018
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 4 6. MATERIALS SUPPLIED 4 7. MATERIALS REQUIRED, NOT SUPPLIED 4 8. TECHNICAL HINTS 5 ASSAY PREPARATION 6 9. REAGENT PREPARATION 6 10. SAMPLE PREPARATION 6 ASSAY PROCEDURE 8 11. ASSAY PROCEDURE 8 DATA ANALYSIS 9 12. TYPICAL DATA 9 RESOURCES 13 13. QUICK ASSAY PROCEDURE 13 14. TROUBLESHOOTING 14 15. NOTES 15
INTRODUCTION INTRODUCTION 1. BACKGROUND Abcam's Lipid Extraction Kit (Chloroform-Free) (ab211044) provides a chloroform-free method of lipid isolation. The recovered organic phase can be dried and resuspended for downstream lipid analysis. This protocol overcomes the difficulties of lower phase extraction and the dangers of using chloroform while resulting in highly efficient lipid extraction. Each kit provides sufficient reagents to isolate up to 50 lipid preparations based on a 25 μl sample size. The term lipids describe a group of naturally occurring molecules that includes monoglycerides, diglycerides, trigylcerides, phospholipids, fatsoluble vitamins, fats and sterols. Lipids define and preserve cellular membrane integrity and play an important role in cellular processes such as membrane trafficking, signal transduction and energy storage. Dysregulation of lipid metabolism is linked to many diseases like cancer, diabetes and heart disease. Traditional lipid extraction is performed using the Folch method, in which lipids are extracted into an organic phase. Isolating lipids from this organic phase is tricky and can result in contamination with material from other phases. These contaminants may have a negative impact on downstream lipid analysis. Furthermore, the Folch method uses chloroform, which is toxic, as the organic phase solvent. ab211044 Lipid Extraction Kit 1
INTRODUCTION 2. ASSAY SUMMARY Prepare sample Add lipid extraction buffer Vortex for 1-2 minutes (For tissue: homogenize, centrifuge, collect supernatant) Agitate for 15 20 minutes at RT. Centrifuge for 5 minutes Collect supernatant and record volume Dry supernatant over night ab211044 Lipid Extraction Kit 2
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. We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet. Reagents should be treated as possible mutagens and should be handle with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components. Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipet by mouth. Do not eat, drink or smoke in the laboratory areas. All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures. 4. STORAGE AND STABILITY Store kit at RT 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 the Materials Supplied section. ab211044 Lipid Extraction Kit 3
GENERAL INFORMATION 5. 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. 6. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) Extraction Buffer 25 ml RT RT Suspension Buffer 2.5 ml RT RT 7. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: Vacuum concentrator Dounce homogenizer Vortex ab211044 Lipid Extraction Kit 4
GENERAL INFORMATION 8. TECHNICAL HINTS This kit is sold based on number of tests. A test simply refers to a single isolation. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures. Avoid foaming or bubbles when mixing. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions Ensure all reagents and solutions are at the appropriate temperature before starting the assay ab211044 Lipid Extraction Kit 5
ASSAY PREPARATION ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening 9.1. Extraction Buffer Provided ready to use. Store at RT and protect from light. 9.2. Suspension Buffer Provided ready to use. Store at RT and protect from light. 10.SAMPLE PREPARATION General Sample Information 10.1. Plasma: 10.1.1. Collect blood in a tube containing an anticoagulant such as citrate, EDTA, or heparin, and mix by inversion. 10.1.2. Centrifuge the blood at 1000 x g at 4 C for 10 minutes. 10.1.3. Collect plasma supernatant without disturbing the white buffy layer. 10.1.4. Samples can be extracted immediately or may be stored in aliquots at -80 C. ab211044 Lipid Extraction Kit 6
ASSAY PREPARATION 10.2. Serum 10.2.1. Collect blood in a tube with no anticoagulant. 10.2.2. Allow the blood to clot at RT for 30 minutes. 10.2.3. Centrifuge at 2500 x g for 20 minutes. 10.2.4. Collect the supernatant without disturbing the white buffy layer. 10.2.5. Samples can be extracted immediately or may be stored in aliquots at -80 C. 10.3. Cultured Cells: 10.3.1. Pellet 1-5 x 10 5 cells by centrifugation at 1000 x g for 5 minutes. 10.3.2. Wash cell pellet once with PBS. 10.3.3. Resuspend pellet in 25-50 µl PBS. 10.3.4. Perform the extraction as described below. 10.4. Tissues 10.4.1. Carefully mince the tissue into small fragments with a scalpel/razor blade and weigh in a 50 ml conical tube. 10.4.2. Use ~10-50 mg of tissue (wet weight) per sample for extraction. 10.4.3. Perform the extraction as described below. ab211044 Lipid Extraction Kit 7
ASSAY PROCEDURE ASSAY PROCEDURE 11.ASSAY PROCEDURE Equilibrate all materials and prepared reagents to correct temperature prior to use. Prepare samples as directed in the previous sections. The protocol below is for a 25 μl sample size. Number of preps per kit will decrease with increasing sample volumes. 11.1. Add 25 μl of sample (or 10-50 mg tissue) to a clean 1.5 ml micro centrifuge tube. 11.2. Add 500 μl of the Extraction Buffer to the sample. 11.3. Immediately vortex for 1-2 minutes using a tube shaker or vortex. NOTE: For tissues, homogenize the tissue pieces in a Dounce homogenizer on ice. Spin the homogenate at 10,000 x g for 5 minutes at 4 C and collect the supernatant. 11.4. Agitate the mixture for 15-20 minutes on an orbital shaker at RT. 11.5. Centrifuge the tube at 10,000 x g for 5 minutes and carefully collect the supernatant, which contains the lipids, and transfer to a clean tube. 11.6. Record the volume. 11.7. Leave the tube containing the supernatant open and dry it in a vacuum concentrator or in a dry 37ºC incubator overnight NOTE: A thin film can be seen after complete drying. The lipid sample can be processed further. If the lipids will be analysed by lipidomics, HPLC or MS, we recommend resuspending the lipid extract in an organic solvent such as butanol or cyclohexane. If the downstream application is analysis of lipids using assay kits (e.g., to quantify cholesterol, triglyceride etc.), we recommend resuspending the lipid extract in 50 μl of Suspension Buffer. Sonicate the lipid extract for 15-20 minutes at 37 C. ab211044 Lipid Extraction Kit 8
DATA ANALYSIS DATA ANALYSIS 12. TYPICAL DATA TYPICAL DATA provided for demonstration purposes only. Analysis of Total Free Fatty Acid, Triglyceride and Cholesterol on Extracted Lipids using Rat Liver Tissue. Figure 1 Comparison of Total Cholesterol using the Folch Method and Abcam s Lipid Extraction (Chloroform-Free) ab211044. The Total Free Cholesterol was quantified using ab204132. ab211044 Lipid Extraction Kit 9
DATA ANALYSIS Figure 2 Comparison of Total Free Fatty Acid using the Folch Method and Abcam s Lipid Extraction (Chloroform-Free) ab211044. The Total Free Cholesterol was quantified using ab65341. Figure 3 Comparison of Total Triglyceride using the Folch Method and Abcam s Lipid Extraction (Chloroform-Free) ab211044. The Total Free Cholesterol was quantified using ab65336. ab211044 Lipid Extraction Kit 10
DATA ANALYSIS Analysis of Total Free Fatty Acid, Triglyceride and Cholesterol on Extracted Lipids using Adipocytes Cell Lysate. Figure 4 Comparison of Total Cholesterol using the Folch Method and Abcam s Lipid Extraction (Chloroform-Free) ab211044. The Total Free Cholesterol was quantified using ab204132. Figure 5 Comparison of Total Free Fatty Acid using the Folch Method and Abcam s Lipid Extraction (Chloroform-Free) ab211044. The Total Free Cholesterol was quantified using ab65341. ab211044 Lipid Extraction Kit 11
DATA ANALYSIS Figure 6 Comparison of Total Triglyceride using the Folch Method and Abcam s Lipid Extraction (Chloroform-Free) ab211044. The Total Free Cholesterol was quantified using ab65336. ab211044 Lipid Extraction Kit 12
RESOURCES RESOURCES 13. QUICK ASSAY PROCEDURE NOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time. NOTE: Get equipment ready. Prepare samples as described in section 10. Extract lipid (note: refer to main protocol for tissue samples) 13.1. Add 25 μl of sample to the tube. 13.2. Add 500 μl of Extraction Buffer to the sample. 13.3. Vortex for 1-2 minutes. 13.4. Agitate the mixture for 15-20 minutes at RT. 13.5. Centrifuge at 10,000 x g for 5 minutes and carefully transfer the supernatant into a clean tube. 13.6. Record the volume. 13.7. Dry supernatant in a vacuum concentrator or a dry 37ºC incubator overnight. ab211044 Lipid Extraction Kit 13
RESOURCES 14.TROUBLESHOOTING Problem Cause Solution Assay not working Sample with erratic readings Lower/ Higher readings in samples and Standards Unanticipated results Use of ice-cold buffer Cells/tissue samples not homogenized completely Samples used after multiple free/ thaw cycles Use of old or inappropriately stored samples Improperly thawed components Incorrect incubation times or temperatures Samples contain interfering substances Buffers must be at room temperature Use Dounce homogenizer, increase number of strokes Aliquot and freeze samples if needed to use multiple times Use fresh samples or store at - 80 C (after snap freeze in liquid nitrogen) till use Thaw all components completely and mix gently before use Verify correct incubation times and temperatures in protocol Troubleshoot if it interferes with the kit ab211044 Lipid Extraction Kit 14
RESOURCES 15.NOTES ab211044 Lipid Extraction Kit 15
RESOURCES ab211044 Lipid Extraction Kit 16
RESOURCES ab211044 Lipid Extraction Kit 17
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