Iipoprotein determination - (Oct/11/2011 )
Im currently trying to separate lipoproteins (HDL, VDL, LDL). Once separated is there anyway to determine what I have separated is that particular type of lipoprotein?
I am not in need to quantify the lipoprotein just prove with some degree that it is either HDL, LDL, VLDL.
I shall then dilute the lipoprotein and place them in to a boyden chambre and analyse to see if any THP-1 differentiated macrophages migrate towards them.
as it shows in the pictures with your procedure, the bands will be prominent and you'll be able to determine which is which by their position in the gradient.
But is there a catagorical way to determine what they are. For example if i took samples off the band and gave them to someone but I did not label them. Could i performa test to one confirm they are lipids and secondly that they are either LDL, HDL etc? for some reason im thing of some sort of electrophoresis procedure!
the papers referenced (and linked) in one of your other threads show electrophoretic identification of the three lipoproteins.
HDL cholesterol precipitating reagent
When serum is reacted with the polyethylene glycol reagent, all the low and very low-density lipoprotein (LDL and VLDL) are precipitated. The HDL fraction remains in the supernatant. The supernatant is then treated as a sample for cholesterol assay.
Glycine-NaOH buffer 0.2 M at pH 10.0 : A) glycine 0.2 M : dissolve in 600 ml dH2O, 15.01 g of glycine and fill up to 1 liter with dH2O; B) NaOH 0.2 M : dissolve 10 g of NaOH in 600 ml dH2O and after fill up to 1 liter with dH2O. For to realize 82 ml of buffer at pH 10.0, mix 50 ml of A with 32 ml of B check pH to verify if is 10.0, and dissolve 15 mM NaN3, while for to realize 123 ml of buffer at pH 10.0, mix 75 ml of A with 48 ml of B check pH to verify if is 10.0, and dissolve 15 mM NaN3.
Precipitating reagent : dissolve 20 g of polyethylene glycol 6000 in glycine-NaOH buffer 0.2 M at pH 10.0. This reagent is ready to use and stored at 2-8 °C protect from light resulting stable tightly capped for 1 year. Once opened the reagent is stable for 2 months at 2-8 °C.
Mix equal amount of serum and precipitating reagent, vortex for 30 seconds and kept at room temperature for 5-10 min., mixing in the meantime a couple of times on vortex. Centrifuged at 3000-4000 rpm (1500-2000 g) for 10 min at 25 °C to obtain a clear supernatant, on which to perform total cholesterol determination.