Native gel lipid binding assay
- 1Yale University
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Abstract
This protocol details how to perform a native gel lipid binding assay to evaluate the number of lipids bound per molecule of 3xFLAG-SHIP164Δ901-1099
Before starting
Before starting
- Transfect and purify protein as previously described [dx.doi.org/10.17504/protocols.io.bp2l61x1kvqe/v1, dx.doi.org/10.17504/protocols.io.x54v9ypjqg3e/v1]
- Purify E-Syt2 as described in doi: 10.1038/nature13269.
- Prepare 10X native running buffer (30 g Tris base, 144 g glycine in 1L). Pre-cool 10X native running buffer and at least 2L of ddH2O to 4ºC.
- Prepare 2X native sample buffer (62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% Bromophenol Blue) and pre-cool to 4ºC.
Set up lipid binding reactions
Set up lipid binding reactions
Quantify protein concentration using a NanoDrop One (Thermo Scientific), where UV absorbances at 280 nm were calculated for 3xFLAG-SHIP164Δ901-1099 and E-Syt2 using the ExPASy ProtParam tool to estimate Abs 0.1% = 0.797 and Abs 0.1% = 0.972, respectively.
Dilute proteins to the indicated concentrations with the same buffer as used for protein purification, to a final volume of 20μL per sample.
Prepare NBD-PE lipid stock by transferring the lipid in chloroform to a borosilicate glass culture tube (12x75mm) using a Hamilton glass syringe, then evaporate the chloroform using a steady stream of N2 gas. Add methanol to a final lipid concentration of 2.705 mM and resuspend lipid by gentle vortexing.
To make the lipid binding reaction samples, add lipid stock quickly to each sample tube to a final working concentration of 0.2 mM. The 0 mM NBD-PE control sample contains the same volume of methanol as the samples with lipid dissolved in methanol. (Note: lipid stock resuspended in methanol must be made immediately before adding to the sample tubes and pipetted quickly to avoid methanol evaporation. Total percentage of methanol in each sample tube should be 5% or less to avoid protein precipitation.)
Gently flick sample tubes to mix, then incubate on ice for 3h in the dark.
Run the native gel
Run the native gel
Add native sample buffer to the tubes to a final concentration of 1X, then load samples onto 4—20% Mini-PROTEAN TGX Precast Gels with 50μL wells (BioRad). Add 1X native running buffer, ice pack, and magnetic stir bar to electrophoresis tank, then keep the electrophoresis tank covered from light. Run the gel for 3h at 200 V while stirring on a magnetic stir plate.
Analyze the native gel
Analyze the native gel
Image the gel for 2 seconds using ImageQuant LAS4000 (GE Healthcare) with the fluorescence method under the Cy2 channel to visualize the NBD fluorescence that comigrated with the protein in the native gel. Then stain the gel with Coomassie Brilliant Blue R (Sigma Aldrich) to visualize total protein using the trans-illumination method.
Calculate band intensities due to NBD fluorescence and Coomassie dye using ImageJ (National Institutes of Health). Obtain the amount of lipid bound to SHIP164 from the standard curve created by the values calculated by the peaks for E-Syt2.