Western blotting of XK and VPS13A
Pietro De Camilli, Chase Amos
Abstract
This protocol describes collection of protein from cultured cells and immunoblotting.
Steps
Cell culture and treatments
Culture K562 or COS-7 (ATCC) at 37°C and 5% CO2, using RPMI for K562 or DMEM for COS-7 containing 10% FBS, 1millimolar (mM) sodium pyruvate, 100 penicillin, 100 streptomycin, 2millimolar (mM) L-glutamine, 1 non-essential amino acids, (all from Gibco) and 2.5 plasmocin (InvivoGen).
For K562 cells treated with hemin, supplement the media with hemin (Sigma Aldrich) dissolved in DMSO to a final concentration of 30micromolar (µM) for 48h 0m 0s .
Cell lysis and sample preparation
Prior to K562 lysis, pellet the cells by centrifuging at 1100rpm,4°C . Resuspend the pellet in PBS, centrifuging and repeating for a total of 3 times.
Resuspend in PBS and centrifuge 1100rpm,4°C (1/3).
Resuspend in PBS and centrifuge 1100rpm,4°C (2/3).
Resuspend in PBS and centrifuge 1100rpm,4°C (3/3).
Prior to lysis of confluent COS-7 cells, aspirate media and wash with PBS 3 times.
Lyse cells with 2% SDS by either resuspending (K562) or adding to culture dish and scraping using a Corning cell-lifter (COS-7). Sonicate lysates using 3x10s pulses with Virsonic 550 (Virtis).
Centrifuge 13300rpm and collect the post-nuclear supernatant in a new Eppendorf tube.
Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).
Prepare samples at desired concentration and add SDS loading buffer to reach a final concentration of 50millimolar (mM) 06.8 Tris, 2Mass / % volume SDS, 0.1Mass / % volume bromophenol blue, 10% (v/v) glycerol, and 1% (v/v) beta-mercaptoethanol.
Boil 95°C for 0h 10m 0s .
Gel electrophoresis and immunoblotting
Prepare gel apparatus with 4-12% Tris Glycine gels (Invitrogen) and Tris-Glycine SDS running buffer.
Load samples into gel and run until dye front reaches bottom (120-150 V).
Remove gel and set up transfer cassette with nitrocellulose membrane.
Transfer at 30 V at 4°C in NuPage transfer buffer (Invitrogen)
Remove nitrocellulose membrane and block membrane with 5% BSA in TBST for 1h 0m 0s at 22Room temperature .
Add primary antibodies at desired concentration in 5% BSA in TBS-T, incubate at 4°C .
Wash membrane with TBST. Repeat a total of 3 times.
Wash membrane for 0h 5m 0s with TBST (1/3).
Wash membrane for 0h 5m 0s with TBST (2/3).
Wash membrane for 0h 5m 0s with TBST (3/3).
Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 5% BSA in TBST for 1h 0m 0s at Room temperature .
Wash membrane with TBST. Repeat a total of 3 times.
Wash membrane for 0h 5m 0s with TBST (1/3).
Wash membrane for 0h 5m 0s with TBST (2/3).
Wash membrane for 0h 5m 0s with TBST (3/3).
Image membranes using a Licor Odyssey Infrared Imager.
