Gfp trap kit

A GFP trap kit (Chromotek) was used to purify GFP-tagged protein from infected cells according to the instructions in the manufacturer's manual. Briefly, 5 × 10⁶ confluent cells were infected with virus expressing a GFP at a multiplicity of infection of 1. After 16 h, the infected cells were harvested and lysed for 20 min on ice in 1 ml radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 1% NP-40, protease inhibitors [Roche]). The cell lysates were centrifuged, and the supernatants were incubated with 15 μl washed GFP-TRAP beads for 30 min at 4°C with rotation. The beads were washed twice and resuspended in loading buffer for analysis by SDS-PAGE.

Schizont, gametocyte and ookinete samples were isolated as described below. WT-GFP or CYC3-GFP samples were then purified using a GFP-Trap kit to immunoprecipitate GFP-fusion protein (Chromotek). After the addition of Laemmli sample buffer, the samples were boiled and an equal concentration of total protein was loaded on a 4–12% SDS-polyacrylamide gel. Samples were subsequently transferred to nitrocellulose membranes (Amersham Biosciences) and immunoblotting performed using the Western Breeze Chemiluminescent Anti-Rabbit kit (Invitrogen) and anti-GFP polyclonal antibody (Invitrogen) at a concentration of 1:1250, according to the manufacturer's instructions.

eYFP-linked receptor constructs were immunoprecipitated from 200 μl cell lysate (5 μg/μl of protein) using the GFP-Trap kit (Chromotek) according to manufacturer's instructions. Immune complexes were washed three times in washing buffer, resuspended in 100 μl 2 × SDS-PAGE sample buffer and incubated at 60 °C for 10 min. Following centrifugation at 2500g for 5 min, 20 μl of immunoprecipitated proteins was resolved by SDS-PAGE on 4% to 12% BisTris (3 (link)) gels. After separation, the proteins were transferred electrophoretically onto nitrocellulose membrane, which was then blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS, 50 mM Tris-Cl, 150 mM NaCl, pH 7.6) for 1 h at room temperature on a rotating shaker. The membrane was then incubated with appropriate primary antibody in 5% BSA powder in TBS supplemented with 0.1% Tween (TBS Tween) overnight at 4 °C on a rotating shaker. Anti-GPR84, anti-pSer²²¹/pSer²²⁴, and anti-pThr²⁶³/pTh^r264 antisera were diluted 1:2000. Subsequently, the membrane was washed (3 × 10 min with TBS-Tween) and incubated for 2 h with anti-rabbit secondary antibody diluted 1:10,000 in 5% BSA in TBS-Tween. After washing (3 × 10 min with TBS-Tween), proteins were detected using Odyssey imaging system according to the manufacturer's instructions.

Immune complexes were collected when 1 mg of cell lysate was immunoprecipitated with anti-Met antibody (CST 3127, 1:50) overnight at 4 ^oC with rotation. Anti-mouse agarose or mouse agarose (both Sigma) were added for 1 h at 4 °C prior to three washes in lysis buffer. Samples were then separated by SDS–PAGE, transferred to a PVDF membrane and immunoblotted. Lysates from cells expressing GFP-tagged proteins were immunoprecipitated using a GFP-Trap Kit (Chromotek) as per manufacturer’s instructions and immunoblotting performed as above. n = 3 and quantitation is shown as mean ± s.d.

The mEGFP-linked receptor construct was immunoprecipitated from 540 µL of cell lysate (3 µg/µL protein) using the GFP-Trap kit (Chromotek) according to manufacturer's instructions. Immune complexes were washed three times in washing buffer, resuspended in 100 µL of 2× SDS-PAGE sample buffer and incubated at 60 °C for 10 min. Following centrifugation at 2,500 × g for 5 min, 40 µL of immunoprecipitated proteins were resolved by SDS-PAGE on 4% to 12% BisTris gels. After separation, immunoblots were carried out as described by Marsango et al. (40 (link)), with the following modifications: Nitrocellulose membranes were blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline (50 mM Tris-Cl, and 150 mM NaCl, at pH 7.6), and anti-pSer²²⁸ M₁ primary and anti-rabbit secondary antibodies were diluted 1:1,000 and 1:10,000, respectively, in 5% BSA in Tris-buffered saline supplemented with 0.1% Tween.