TITLE: Evaluating GPNMB ACD mutants by Western Blotting and immunofluorescence.
AUTHORS: Erin Bogacki, Patrick Lewis, sherbst

[DESCRIPTION]
This protocol describes the evaluation of cellular processing of GPNMB mutants by Western Blotting and Immunofluorescent imaging in a HEK293 overexpression model. 

[STEPS]
SECTION: Seed cells
1. Seed HEK293 cells. 
A) For Western Blotting, we recommend seeding 2.5 x 10^5 cells per well of a 12-well culture plate.
B) For Immunofluorescence, we recommend seeding 1.2 x 10^5 cells per well of a 24-well culture plate. Seed cells on Poly-D-Lysine coated coverslips. 
Note: we routinely culture HEK293T cells in DMEM containing 10% FCS. 
Incubate in a tissue culture incubator   .

SECTION: HEK 293 cell transfection
2. This protocol uses a DNA: Fugene ratio of 1:3. Prepare the transfection complexes as follows:

 

SECTION: HEK 293 cell transfection
2.1. Add the required amount of plasmid DNA to serum-free DMEM. Mix briefly.
SECTION: HEK 293 cell transfection
2.2. Add the required amount of Fugene HD Transfection Reagent. 
SECTION: HEK 293 cell transfection
2.3. Vortex and incubate for 10 min at Room temperature .

SECTION: HEK 293 cell transfection
2.4. In the meantime, change the medium on the cells to fresh DMEM containing 10 % FCS
SECTION: HEK 293 cell transfection
2.5. Add 50 µL per 24-well or 100 µL per 12-well drop-wise to the cells and incubate   

SECTION: Western Blotting
3. This section describes the sample preparation and analysis for Western Blotting. 
SECTION: Western Blotting
3.1. Wash the cells gentle with PBS
SECTION: Western Blotting
3.2. Immediately add 100 µL  per well of ice-cold cell lysis buffer and place cells on ice.

SECTION: Western Blotting
3.3. Scrape cells with a cell scraper and harvest cell lysate into a 1.5 ml Eppendorf tube. 
SECTION: Western Blotting
3.4. Incubate the cells on ice for 10 min , vortex occasionally

SECTION: Western Blotting
3.5. Clear the cell lysate by spinning down at 16200 x g, 4 °C for 15 min 

SECTION: Western Blotting
3.6. Transfer the post-nuclear supernatant to a fresh 1.5 ml Eppendorf tube and store at -20 °C .

SECTION: Western Blotting
3.7. Prepare the cell lysates for Western Blotting by adding LDS sample buffer and denaturing agent and denature the samples at 80 °C for 8 min.

SECTION: Western Blotting
3.8. Run samples on 4-12 % Bis-Tris SDS-page. (approx. 35 min at 160 V const.)

SECTION: Western Blotting
3.9. Transfer proteins onto a PVDF membrane using the Turbo transfer system (BioRad) or similar.
SECTION: Western Blotting
3.10. Block membranes in 5% milk/TBS-T for 60 min at Room temperature .

SECTION: Western Blotting
3.11. Incubate the membranes with primary antibodies at 4 °C   .
 


SECTION: Western Blotting
3.12. Wash the plots in TBS-T for 5 min  at Room temperature. Repeat this step twice for a total of three washes.  

SECTION: Western Blotting
3.13. Dilute the secondary antibody in 5% milk/TBS-T and incubate the membranes with secondary antibodies at 4 Room temperature for 45 min.
 

SECTION: Western Blotting
3.14. Develop the blots using an appropriate developer. Full-length GPNMB-EGFP is detected as a double band at ~125 kDa. A cleaved C-terminal GPNMB fragment can be detected with the anti-GFP antibody at ~35 kDa. 
SECTION: Immunofluorescence
4. Gently wash coverslips with PBS and fix in 4 % PFA/PBS for 15 min min.

SECTION: Immunofluorescence
4.1. Gently wash the cells with PBS. Replace the PBS and add the permeabilisation/blocking. Incubate coverslips for a minimum of 20 min min at Room temperature .

SECTION: Immunofluorescence
4.2. In the meantime, place a piece of Parafilm onto your bench and label if required. This will act as a flat surface to stain the coverslips on. 
SECTION: Immunofluorescence
4.3. Prepare the antibody staining solution in Blocking and staining buffer. Find a suggestion of antibodies for counterstaining below:
 

SECTION: Immunofluorescence
4.4. Pipette a 45 µL drop of the antibody staining solution onto the Parafilm and invert the coverslip onto the staining solution so that the cells face downwards. 

SECTION: Immunofluorescence
4.5. Incubate for 60 min hr in the dark.

SECTION: Immunofluorescence
4.6. Wash the coverslips three times with PBS.
SECTION: Immunofluorescence
4.7. Prepare a staining solution containing the secondary antibody: 
 

SECTION: Immunofluorescence
4.8. Pipette a 45 µL drop of the antibody staining solution onto the Parafilm and invert the coverslip onto the staining solution so that the cells face downwards. 
SECTION: Immunofluorescence
4.9. Incubate for 45 min min in the dark.

SECTION: Immunofluorescence
4.10. Wash the coverslips once in PBS, stain with DAPI (or other nuclear stain), and mount onto glass slides.
SECTION: Immunofluorescence
4.11. GPNMB can be observed predominantly at the trans-Golgi network but can also be seen at lysosomal compartments.