Anti mouse hrp conjugate

The 2C11 affinity purified anti-PI(4,5)P2 antibody was from Echelon (Salt Lake City, UT, USA) product number Z-P045, lot number XCM042523-23, and used at 1:200 dilution in blocking buffer (see IF methods for blocking buffer). The affinity-purified anti-PI(3,4,5)P3 antibody was also from Echelon product number Z-G345, lot number ML120516-23, and used at 1:200 dilution in blocking buffer. The anti-3X FLAG M2 monoclonal antibody was from Sigma (St. Louis) and used at 1:1000 dilution in blocking buffer. For colocalization of FLAG and PIP2 in the same chamber, mouse FLAG-primary M2 antibodies were detected with 1:1000 PBS-diluted Invitrogen Alexa-Fluor 594 goat anti-mouse-IgG1, lot number 2566384, while 2C11 anti-PIP2 primary antibodies were detected with 1:2000 PBS-diluted Invitrogen Alexa-Fluor 488 goat anti-mouse-IgM, lot number 1896382. The anti-actin antibody was from Cell-Signaling (product number 8H10D10) and was used at 1:5000 dilution in 1X TBST. The secondary antibody in the Westerns was Promega anti-mouse HRP conjugate (product number W402B) and was used at 1:10,000 dilution in 1X TBST.

Three micrograms of proteins from the whole cell lysates or cytoplasmic/periplasmic fractions were mixed with 3 × SDS Blue Loading Buffer (New England Biolabs Inc.), denatured at 95°C for 10 min, and then loaded in 12% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) for electrophoresis at 80–120 V for 90 min. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad) at 25 V for 30 min using a Trans-Blot semidry transfer cell (Bio-Rad) in transfer buffer (14.41 g/L glycine, 3.03 g/L Tris base, 0.075 g/L SDS, and 20% methanol). Detection of GFP was achieved through an anti-GFP antibody (Sigma-Aldrich, G6539) at a 1:2,000 dilution and an anti-mouse horseradish peroxidase (HRP) conjugate (Promega, W4021) at a 1:2,500 dilution. Detection of 6 × His was achieved through an anti-His antibody (ThermoFisher Scientific, MA1–135) at a 1:2,500 dilution and an anti-mouse-HRP conjugate (Promega, W4021) at a 1:2,500 dilution. Protein samples were visualized by SYPRO Ruby (Thermo Fisher Scientific) staining in parallel to show that the protein amount loaded in each lane is equivalent. The signals were developed using Clarity Western enhanced chemiluminescence substrate (Bio-Rad). Quantitation of Western blot band intensities was done using the CLIQS software (Total Lab).

Protein was transferred to PVDF-membrane (GE Healthcare, Buckinghamshire, UK) by wet-western-blotting, with electrophoresis for 1 h at 80 V, 300 A. The PVDF membrane was then immersed in blocking solution (2.5% (w/v) skimmed milk powder in 50 mL 1× PBS-Tween20 (0.1%) and incubated at 4 °C overnight. The following day the membrane was washed 3× for 5 min with 1× PBS-Tween20 (0.1%) before incubation with primary antibody (3.5 μL anti-6-His (Life Technologies, CA, USA) in 20 mL 1× PBS-Tween20 (0.1%), 3% (w/v) BSA) for 1 h at room temperature. Membranes were then washed 3× for 5 min with 1× PBS-Tween20 (0.1%) before incubation with secondary antibody [4 μL anti-Mouse HRP conjugate (Promega, WI, USA) in 20 mL 1× PBS-Tween20 (0.1%)] for 1 h at room temperature. Finally, membranes were washed for 3× for 5 min with 1× PBS-Tween20 (0.1%). Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (BioRad, Herts, UK) following manufacturer’s instructions. Bands were visualised using BioRad Gel-doc chemiluminescence imager and associated software. Comparative densitometry of band-intensities was also carried out on a BioRad Gel-doc imager with an Image Lab™ Software 4.1.