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Iipoprotein determination

lipoprotein electrophoresis TLC HPLC

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4 replies to this topic

#1 swizla21

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Posted 11 October 2011 - 03:27 PM

Hi,

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.

Riz

#2 mdfenko

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Posted 12 October 2011 - 11:38 AM

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.
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#3 swizla21

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Posted 12 October 2011 - 05:07 PM

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!

#4 mdfenko

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Posted 13 October 2011 - 05:09 AM

the papers referenced (and linked) in one of your other threads show electrophoretic identification of the three lipoproteins.

Edited by mdfenko, 13 October 2011 - 05:15 AM.

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#5 Zagami Francesco

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Posted 08 March 2015 - 10:45 AM

HDL cholesterol precipitating reagent

Principle

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.

Reagents

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.

Procedure

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.







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