TITLE: Western blotting of XK and VPS13A
AUTHORS: Chase Amos, Pietro De Camilli

[DESCRIPTION]
This protocol describes collection of protein from cultured cells and immunoblotting.

[STEPS]
SECTION: Cell culture and treatments
1. Culture K562 or COS-7 (ATCC) at 37 °C  and 5% CO2, using RPMI for K562 or DMEM for COS-7 containing 10% FBS, 1 millimolar (mM) sodium pyruvate, 100 U/mL penicillin, 100 μg/mL  streptomycin, 2 millimolar (mM)  L-glutamine, 1 x  non-essential amino acids, (all from Gibco) and 2.5 μg/mL plasmocin (InvivoGen).
SECTION: Cell lysis and sample preparation
3. Prior to K562 lysis, pellet the cells by centrifuging at 1100 rpm, 5 min, 4 °C . Resuspend the pellet in PBS, centrifuging and repeating for a total of 3 times.
SECTION: Cell lysis and sample preparation
5. 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).
SECTION: Cell lysis and sample preparation
6. Centrifuge 13300 rpm, 10 min  and collect the post-nuclear supernatant in a new Eppendorf tube.
SECTION: Cell lysis and sample preparation
7. Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).
SECTION: Cell lysis and sample preparation
8. Prepare samples at desired concentration and add SDS loading buffer to reach a final concentration of 50 millimolar (mM)   pH 06.8  Tris, 2 Mass / % volume  SDS,  0.1 Mass / % volume bromophenol blue,  10 % (v/v) glycerol, and 1 % (v/v) beta-mercaptoethanol. 

SECTION: Gel electrophoresis and immunoblotting
10. Prepare gel apparatus with 4-12% Tris Glycine gels (Invitrogen) and Tris-Glycine SDS running buffer.
SECTION: Gel electrophoresis and immunoblotting
11. Load samples into gel and run until dye front reaches bottom (120-150 V).
SECTION: Gel electrophoresis and immunoblotting
12. Remove gel and set up transfer cassette with nitrocellulose membrane.
SECTION: Gel electrophoresis and immunoblotting
13. Transfer at 30 V      at 4 °C in NuPage transfer buffer (Invitrogen)

SECTION: Gel electrophoresis and immunoblotting
14. Remove nitrocellulose membrane and block membrane with 5% BSA in TBST for 60 min  at 22 Room temperature .

SECTION: Gel electrophoresis and immunoblotting
16. Wash membrane with TBST. Repeat a total of 3 times.
SECTION: Gel electrophoresis and immunoblotting
16.1. Wash membrane for 5 min with TBST (1/3).

SECTION: Gel electrophoresis and immunoblotting
16.2. Wash membrane for 5 min with TBST (2/3).
SECTION: Gel electrophoresis and immunoblotting
16.3. Wash membrane for 5 min with TBST (3/3).
SECTION: Gel electrophoresis and immunoblotting
17. Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 5% BSA in TBST for 60 min  at Room temperature .

SECTION: Gel electrophoresis and immunoblotting
19. Image membranes using a Licor Odyssey Infrared Imager.
SECTION: Cell culture and treatments
2. For K562 cells treated with hemin, supplement the media with hemin (Sigma Aldrich) dissolved in DMSO to a final concentration of 30 micromolar (µM)  for 2880 min .
SECTION: Cell lysis and sample preparation
4. Prior to lysis of confluent COS-7 cells, aspirate media and wash with PBS 3 times.
SECTION: Cell lysis and sample preparation
9. Boil 95 °C  for 10 min .
SECTION: Gel electrophoresis and immunoblotting
18. Wash membrane with TBST. Repeat a total of 3 times.
SECTION: Gel electrophoresis and immunoblotting
18.1. Wash membrane for 5 min with TBST (1/3).
SECTION: Gel electrophoresis and immunoblotting
18.2. Wash membrane for 5 min with TBST (2/3).
SECTION: Gel electrophoresis and immunoblotting
18.3. Wash membrane for 5 min with TBST (3/3).
SECTION: Cell lysis and sample preparation
3.1. Resuspend in PBS and centrifuge 1100 rpm, 5 min, 4 °C  (1/3).

SECTION: Cell lysis and sample preparation
3.2. Resuspend in PBS and centrifuge 1100 rpm, 5 min, 4 °C  (2/3).

SECTION: Cell lysis and sample preparation
3.3. Resuspend in PBS and centrifuge 1100 rpm, 5 min, 4 °C  (3/3).

SECTION: Gel electrophoresis and immunoblotting
15. Add primary antibodies at desired concentration in 5% BSA in TBS-T, incubate    at 4 °C .