LDL/VLDL Purification Kit (Ultracentrifugation Free)

Produktnummer ABIN2345117

Produktdetails

Applikation: Purification (Purif)

Verwendungszweck: The LDL/VLDL Purification Kit uses Dextran Sulfate to selectively precipitate LDL/VLDL from plasma or serum.

Proben: Plasma, Serum

Produktmerkmale: The kit allows for the purification of LDL/VLDL without the need for ultracentrifugation. The 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 for human samples (expected yield will vary by species).

Bestandteile:

1. Dextran Solution : One 0.6 mL vial
2. Precipitation Solution A : One 6 mL amber bottle
3. Bicarbonate Solution : One 4 mL bottle
4. 10X Precipitation Solution B : One 10 mL bottle
5. NaCl Solution : One 6 mL bottle containing 5% NaCl
6. 10X Precipitation Solution C : One 20 mL bottle
7. Dextran Removal Solution : One 1.6 mL vial

Benötigtes Material:

1. Serum or Plasma Samples
2. PBS
3. Microcentrifuge or Centrifuge
4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips

Anwendungsinformationen

Applikationshinweise: 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. 3 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. 3. Discard the supernatant. Use the remaining pellet which contains LDL and VLDL for section II below. 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. 4

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Informationen zur Lagerung: Upon receipt store Dextran Removal Solution at room temperature. Store all other components at 4°C.

Referenzen

Ito, Ito, Suzuki, Yahata, Ikeda, Hamaoka: "The Application of a Modified d-ROMs Test for Measurement of Oxidative Stress and Oxidized High-Density Lipoprotein." in: International journal of molecular sciences, Vol. 18, Issue 2, (2017) (PubMed).

Antigendetails

Hintergrund: 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).