Project description:We used a 3-step protocol that separates lipid-associated proteins based on their buoyant density, size and charge. All steps were conducted either on ice or at 4C. Step 1 involved a sucrose density gradient centrifugation in which ca. 100 postnatal day 4 rat brains were homogenized in buffer A (5 mM MES, pH 7.0, 0.3 M sucrose, 1 mM EDTA) containing protease inhibitor cocktail. The homogenate was centrifuged at 1,000xg for 15 min. Post-nuclear supernatant (PNS) was lysed with 10 volumes of 5 mM MES pH 7.0, 1 mM EDTA and incubated for 30 min at 4C. The lysed PNS was then centrifuged at 100,000 x g for 1 h. The pellet was resuspended in buffer A and loaded on top of a discontinuous sucrose gradient of 0.8, 1.2 and 2.0 M. The gradient was spun for 3 h at 270,000 x g. The material between 0.3 and 0.8 M sucrose was removed, diluted with 5 mM MES, pH 7.0, 1 mM EDTA to a final sucrose concentration of 0.3 M, and centrifuged at 100,000 x g for 1 h. The pellet was further resuspended in a final volume of 2 mL of PBS with protease inhibitors. In step 2, this material was applied to a gel filtration column packed with Sephacryl S-1000 Superfine matrix. The dimensions of the column were 1.5 cm wide and approximately 1.5 m tall. Material flowed through the column by gravity with elution by 1x phosphate buffered saline (PBS, pH 7.4). The flow rate was ca. 0.5 mL/min, and a BioRad Model 2110 fraction collector was used to collect 4 mL per fraction. For the final step 3 of the protocol, the four Sephacryl S-1000 fractions most highly enriched in Piccolo and Bassoon as determined by WB were pooled and applied to a column packed with Q Sepharose Fast Flow. The dimensions of this column were diameter of 2.5 cm, height of approximately 12 cm with a bed volume of 60 mL packed Q Sepharose, and the flow rate using the 2110 fraction collector was 3 mL/min and 10 mL fractions were collected. The final elution profile (all buffers in 20 mM Tris pH 7.0) was 200 mL of 150 mM NaCl to wash, then stepwise elution of 120 mL buffer with the following NaCl concentrations: 210 mM, 290 mM, 350 mM, and 1000 mM. About 40 mL of the 210 mM, 290 mM (Piccolo and Bassoon enriched), and 350 mM NaCl fractions were concentrated to about 1 mL using Amicon Centricon Plus-20 30 kDa cutoff concentrators. The final recovery was 7.5 ug in the 210 mM fraction, and 16 ug in each of the 290 mM and 350 mM fractions. The fractions were precipitated with tricholoroacetic acid, and the protein pellet was stored at -20C until further use.
The second approach to identify proteins associating with Piccolo and Bassoon was an anti-Piccolo IP. The starting material was 10 mg of the fraction between 0.3 and 0.8 M sucrose after density centrifugation (step 1 above). This material was diluted to 0.3 M sucrose with 5 mM MES, pH 7.0 and centrifuged at 100,000 x g for 1 h. The pellet was resuspended and incubated for 1 h with 4 mL of solubilization buffer (5 mM MES, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail). Lysate was cleared by centrifugation at 100,000 x g for 1 h. One mL of supernatant was incubated with 2.5 ug anti-Piccolo rabbit antibody or 2.5 ug rabbit IgG for 4 h. A 100 uL Protein A agarose slurry (Roche) was then added and further incubated overnight. Material was transferred to a small column and washed with 20 mL solubilization buffer. Proteins were eluted twice with 200 uL 50 mM Tris, pH 6.5, 100 mM DTT, 2% SDS. This material was then extracted with 4 volumes methanol and 1-volume chloroform. This mixture was combined with water, centrifuged for 90 sec at 16,000 x g. The aqueous phase was removed and extracted again with 3 volumes of methanol. This precipitated protein was collected by centrifugation for 90 sec at 16,000 x g, methanol removed, and the speed-vacuum was used to dry the protein pellet.
The 210 mM, 290 mM, and 350 mM NaCl fractions from the Mono-Q column protocol and the eluates (both IgG alone and anti-Piccolo IPs) from two separate IP experiments were subjected to Multi-dimensional Protein Identification technology.
2016-07-11 | MSV000079897 | MassIVE
