One ml of radiolabeled virions was applied over 10 ml of 5 to 40% sucrose gradient in PBS and ultracentrifuged at 27,600 rpm for 18 h at 4°C in a SW44Ti rotor (Beckman). Nine equal fractions were collected from the top and an aliquot of each fraction was removed for density measurement by hand held refractometer (R-5000, Atago, U.S.A). The remainder of each fraction was diluted with 3 volumes of PBS and centrifuged at 39,500 rpm for 16 h at 4°C in a SW55 rotor. The pellet was resuspended in NuPAGE sample buffer and analyzed by SDS-PAGE.
Sw44ti rotor
Manufactured by Beckman Coulter
The SW44Ti rotor is a high-speed ultracentrifuge rotor designed for Beckman Coulter's line of ultracentrifuges. It is capable of reaching speeds up to 44,000 revolutions per minute, enabling the separation and concentration of small particles and molecules within a sample.
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Lab products found in correlation
4 protocols using sw44ti rotor
1
Sucrose Gradient Purification of Radiolabeled Virions
2
Viral Particle Density Profiling
3
Cholesterol Quantification of Purified Virus
4
Astrocyte-Derived Lipoprotein Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
APOE3 and APOE4 astrocytes were grown to confluency, washed once with MEM, and incubated in MEM for 3 days. Astrocyte‐conditioned medium (ACM) was spun at 3,000 g for 3 min to remove debris and analyzed first by Western blot against ApoE to confirm the presence of lipoproteins. Prior to all experiments, we measured the amount of ApoE present in the ACM by dot blot (Bio‐Rad Bio‐Dot™ Apparatus) (it typically contained more ApoE3 than ApoE4, consistent with the finding that the concentration of ApoE3 in the brain homogenates23 and in the interstitial fluid of human ApoE knock‐in mice 24 is ~50% greater than that of ApoE4) and then added the ACM on an “equal ApoE” basis, by adding MEM to the ApoE3 ACM as appropriate.
Density ultracentrifugation was used to prepare enriched fractions of lipoproteins70 . Briefly, 100 ml ACM was concentrated to ~4 ml using Centriprep 10K filters (EMD Millipore). Four milliliters of ACM concentrate was layered on top of 3 ml 1.1 g/ml, 3 ml 1.2 g/ml, 2 ml 1.3 g/ml discontinuous sucrose gradient. Samples were centrifuged at 160,000 g in a Sw44‐Ti rotor (Beckman) for 48 h at 4°C. One‐millilitre fractions were removed from the top of the gradient, and ApoE content was analyzed by Western blot.
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APOE3 and APOE4 astrocytes were grown to confluency, washed once with MEM, and incubated in MEM for 3 days. Astrocyte‐conditioned medium (ACM) was spun at 3,000 g for 3 min to remove debris and analyzed first by Western blot against ApoE to confirm the presence of lipoproteins. Prior to all experiments, we measured the amount of ApoE present in the ACM by dot blot (Bio‐Rad Bio‐Dot™ Apparatus) (it typically contained more ApoE3 than ApoE4, consistent with the finding that the concentration of ApoE3 in the brain homogenates
Density ultracentrifugation was used to prepare enriched fractions of lipoproteins
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