Mouse anti halotag

Cells were lysed with 2 × Laemmli buffer [0.1 mm Tris–HCl (pH 6.8), 4% SDS, 20% Glycerol, 1.7 m 2‐mercaptoethanol] or with loading buffer [62.5 mm Tris–HCl (pH 6.8), 2% SDS, 10% glycerol and 41.6 mm dithiothreitol] and sonicated for 20 s, and incubated overnight at 55 °C. Samples were separated using an 8% acrylamide gel and transferred onto a polyvinylidene difluoride membrane (Merck Millipore Ltd, Billerica, MA, USA) and incubated overnight at 4 °C with mouse anti‐HaloTag (1 : 1000, Cat: G9211; Promega), rabbit anti‐WFS1 (1 : 1000) [29 (link)], or mouse anti‐α‐tubulin (1 : 2000, Cat: T6074; Sigma, Billerica, MA, USA). After washing with PBST (PBS containing 0.1% Tween20), the membrane was probed with a horseradish peroxidase‐conjugated antibody. The membrane was scanned using FUSION‐SOLO.4S.WL (M&S Instruments Inc., Tokyo, Japan) after reacting with Luminata Forte Western HRP Substrate (Merck Millipore).

The following antibodies were used in this study: rabbit anti-CHC17 (one made in house and one purchased from ab172958; Abcam); mouse anti-CHC17 (X22; Abcam); rabbit anti-AP1G1 (made in house); rabbit anti-GFP (one a gift from Matthew Seaman, CIMR, Cambridge, UK, and one purchased from EPR14104; Abcam); mouse anti-HaloTag (G9211; Promega); rabbit anti-CIMPR (made in house); mouse anti-AP-2 α subunits (AC1-M11; Abcam); and mouse anti-GGA2 (a gift from Doug Brooks, University of South Australia, Australia).
In addition, a new antiserum was produced against mRuby2, which was not recognized by antibodies against other red fluorescent proteins because it comes from a different organism (Entacmaea quadricolor instead of Discosoma). The entire coding sequence was expressed as a GST fusion protein, affinity-purified using glutathione-Sepharose (GE Healthcare), and used to raise antibodies in rabbits following our established protocol (Page et al., 1999 (link)). The titer of the serum was found to be extremely high, producing very strong labeling in both immunofluorescence and Western blotting experiments when it was diluted 1:10,000, so it was not affinity purified.