LDL/VLDL and HDL Purification Kit (Ultracentrifugation Free)
- Purification (Purif)
- The LDL/VLDL and HDL Purification Kit uses Dextran Sulfate to selectively and separately precipitate LDL/VLDL and HDL fractions.
- Plasma, Serum
- The kit allows for the purification of LDL/VLDL and/or HDL without the need for ultracentrifugation. The various lipoprotein particles are highly purified through a series of precipitation and low speed centrifugation steps. Each kit provides sufficient reagents to perform up to 10 preps, and each preparation can purify up to 10 mL of serum or plasma samples with a yield of ~600 μg of LDL/VLDL per mL and ~4500 μg of HDL per mL for human samples (expected yield will vary by species).
- Dextran Solution : One 8 mL bottle
- Precipitation Solution A : One 30 mL amber bottle
- Bicarbonate Solution : One 4 mL bottle 3
- 10X Precipitation Solution B : One 10 mL bottle
- Tris Solution : One 50 mL bottle containing 20 mM Tris, pH 7.5
- NaCl Solution : One 6 mL bottle containing 5% NaCl
- 10X Precipitation Solution C : One 20 mL bottle
- Dextran Removal Solution : One 10 mL bottle
- 5X HDL Wash Solution : One 12 mL bottle
- Benötigtes Material
- Serum or Plasma Samples
- Microcentrifuge or Centrifuge
- 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
Tumor Necrosis Factor alpha (TNF alpha) ELISA Kit
TNF alpha Reaktivität: Human AA 77-233 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Plasma (EDTA), Plasma (citrate), Plasma (heparin), SerumBrain-Derived Neurotrophic Factor (BDNF) ELISA Kit
BDNF Reaktivität: Maus AA 129-247 Colorimetric Sandwich ELISA 31.2-2000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma (EDTA), Plasma (citrate), Plasma (heparin), SerumChemokine (C-C Motif) Ligand 2 (CCL2) ELISA Kit
CCL2 Reaktivität: Human Colorimetric Sandwich ELISA 31.25-2000 pg/mL Cell Culture Supernatant, Cerebrospinal Fluid, Plasma, Saliva, Serum, Tissue Homogenate, Urine
- Optimal working dilution should be determined by the investigator.
- Aufbereitung der Reagenzien
1X Precipitation Solution B: Dilute the 10X Precipitation Solution B to 1X with deionized water. Stir to homogeneity. Store unused solution at 4 °C. 1X Precipitation Solution C: Dilute the 10X Precipitation Solution C to 1X with deionized water. Stir to homogeneity. Store unused solution at 4 °C. HDL Resuspension Buffer: Dilute Dextran Solution 1:100 and Precipitation Solution A 1:10 in Tris Solution. For example, add 50 L of Dextran Solution and 0.5 mL of Precipitation Solution A to 4.45 mL of Tris Solution. Stir to homogeneity. Prepare only enough for immediate use and do not store unused buffer. 1X HDL Wash Solution: Dilute the 5X HDL Wash Solution to 1X with deionized water. Stir to homogeneity. Store unused solution at 4 °C. Purification Protocol Note: The purification protocol below is written for a 10 mL sample size. For smaller sample volumes, scale down each step proportionally. I. Dextran Precipitation 1. To 10 mL of serum or plasma on ice, add 50 μL of Dextran Solution and 500 μL of Precipitation Solution A. Incubate 5 minutes on ice. 2. Spin at 6000 x g 10 minutes at 4 °C. 4 3. Remove the supernatant, which contains HDL, for use in section III below. Use the remaining pellet which contains LDL and VLDL for section II below. Note: The HDL-containing supernatant may be stored at 4 °C for future processing. II. LDL/VLDL Purification 1. Resuspend the pellet from section I above with 400 μL of Bicarbonate Solution and spin at 6000 x g 10 minutes at 4 °C. 2. Transfer the supernatant to a new tube. Discard the pellet. 3. Add 10 mL of 1X Precipitation Solution B to the supernatant. Mix thoroughly by pipetting up and down. 4. Spin at 6000 x g for 10 minutes at 4 °C. 5. Discard the supernatant and resuspend the pellet with 200 μL of NaCl Solution. 6. Add 10 mL of 1X Precipitation Solution C. Mix thoroughly by pipetting up and down. 7. Spin at 6000 x g for 10 minutes at 4 °C. 8. Repeat steps 5-7. 9. Resuspend the pellet in 200 μL of NaCl Solution (final volume about 500 μL). 10. Add 80 μL of Dextran Removal Solution. Mix thoroughly by pipetting up and down and incubate 1 hour at 4 °C. 11. Spin at 6000 x g for 10 minutes at 4 °C. 12. Recover the supernatant (purified LDL/VLDL) and transfer to a new tube. 13. Dialyze the purified LDL/VLDL in PBS and determine the protein concentration. III. HDL Purification 1. To 10 mL of supernatant from section I above, add 600 μL of Dextran Solution and 1.5 mL of Precipitation Solution A. Incubate for 2 hours at room temperature. 2. Spin 18,000-20,000 x g for 30 minutes at 4 °C. 3. Discard supernatant and resuspend pellet in 5 mL of HDL Resuspension Buffer (see Preparation of Reagents Section). Mix thoroughly by pipetting up and down. 4. Spin 6000 x g for 10 minutes at 4 °C. 5. Discard supernatant and resuspend pellet in 6 mL of 1X HDL Wash Solution (see Preparation of Reagents Section). 6. Shake for 30 minutes at 4 °C. Shaking speed should be sufficient to dissolve pellet, but not so vigorous that bubbles form. 7. Spin 6000 x g for 10 minutes at 4 °C. 8. Transfer the supernatant to a new tube and add 900 μL of Dextran Removal Solution. Mix thoroughly by pipetting up and down. 5 9. Incubate for 1 hour at 4 °C. 10. Spin 6000 x g for 10 minutes at 4 °C. 11. Transfer the supernatant (containing purified HDL) to a new tube. 12. Dialyze the purified HDL in PBS and determine the protein concentration.
- Nur für Forschungszwecke einsetzbar
- 4 °C
- Informationen zur Lagerung
- Upon receipt store Dextran Removal Solution at room temperature. Store all other components at 4°C.
Genetic dissection in a mouse model reveals interactions between carotenoids and lipid metabolism." in: Journal of lipid research, Vol. 57, Issue 9, pp. 1684-95, (2016) (PubMed).
- Genetic dissection in a mouse model reveals interactions between carotenoids and lipid metabolism." in: Journal of lipid research, Vol. 57, Issue 9, pp. 1684-95, (2016) (PubMed).
- Lipoproteins are submicroscopic particles composed of lipid and protein held together by noncovalent forces. Their general structure is that of a putative spheroidal microemulsion formed from an outer layer of phospholipids, unesterified cholesterol, and proteins, with a core of neutral lipids, predominately cholesteryl esters and triacylglycerols (TAG). Very low density lipoprotein (VLDL), a spherical particle with a diameter of 30-100 nm, is the major plasma vehicle for TAG and is the precursor to Low density lipoprotein (LDL). Each VLDL contains one molecule of a hydrophobic protein known as apolipoprotein B-100 (Apo B), as well as multiple copies of apolipoprotein E and apolipoprotein C (Figure 1 left). LDL is the major transport protein for cholesterol in human plasma. LDL, like VLDL, is also a spherical particle with a diameter of 20-25 nm. Each LDL particle contains cholesteryl esters in its core which are surrounded by a hydrophilic coat composed of phospholipids, cholesterol, and one molecule apolipoprotein B-100 (Figure 1 right). Figure 1: Structure of VLDL (left) and LDL (right). High Density Lipoprotein (HDL) is also a spherical particle with diameter of about 10 nm (Figure 2). HDL contains the Apoliprotein AI and AII molecules. HDL and LDL cholesterol levels in the blood are important indicators of many disease states. High blood levels of LDLs are associated with health problems and cardiovascular disease. For this reason, LDL is often referred to as the "bad cholesterol." LDL particles that accumulate within arteries can form plaques over time, which can increase chances of a stroke, heart attack, or vascular disease. HDL particles are able to remove cholesterol from within arteries and transport it back to the liver for re-utilization or excretion, which is the main reason why the cholesterol carried within HDL particles is sometimes called "good cholesterol." Monitoring circulatory levels of different lipoproteins is critical to the diagnosis of lipid transport disorders such as atherosclerosis. 2 Figure 2: Structure of HDL.