Jan 15, 2024

Public workspaceEvaluating GPNMB ACD mutants by Western Blotting and immunofluorescence.

DOI
dx.doi.org/10.17504/protocols.io.4r3l22yz4l1y/v1
  • 1NIH;
  • 2Royal Veterinary College
sherbst
Open access
DOI: dx.doi.org/10.17504/protocols.io.4r3l22yz4l1y/v1
Protocol Citation: Erin Bogacki, Patrick Lewis, sherbst 2024. Evaluating GPNMB ACD mutants by Western Blotting and immunofluorescence.. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22yz4l1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 13, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 90843
Keywords: ASAPCRN, GPNMB, Western Blotting, Immunofluorescence, HEK293
Abstract
This protocol describes the evaluation of cellular processing of GPNMB mutants by Western Blotting and Immunofluorescent imaging in a HEK293 overexpression model.
Materials
General

  • HEK293T cells (ATCC CRL-3216)
  • Fugene HD transfection reagent (E2311, Promega)
  • pcDNA3.1-GPNMB-EGFP Download pcDNA3.1-GPNMB-EGFP.pngpcDNA3.1-GPNMB-EGFP.png104KB
  • PBS, pH 7.4: #14190250, ThermoFisher Scientific

Western Blotting

  • Lysis buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% (v/v) Triton X-100
NOTE: add protease and phosphatase inhibitors fresh each time (eg Halt Protease and Phosphatase Inhibitor Cocktail (100X), #78440, ThermoFisher Scientific)
  • Loading buffer: NuPAGE LDS sample buffer (#NP0007, ThermoFisher Scientific)
  • Sample reducing agent: NuPAGE sample reducing agent (#NP0009, ThermoFisher Scientific)
  • 4-12% Bis-Tris NuPAGE gels (eg #NP0321BOX, ThermoFisher Scientific)
  • SDS-PAGE running buffer: MES running buffer (#NP0002, ThermoFisher Scientific)
  • Trans-Blot‱ Turbo‱ PVDF Transfer Packs: eg #1704157, BioRad
  • TBS-T: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20.
  • Blocking and antibody dilution buffer: 5 % (w/v) non-fat milk powder in TBS-T
  • Primary and secondary antibodies (see table 1 & 2 for antibody suggestions)

Immunofluorescence

  • 4 % (v/v) PFA/PBS: Dilute 16 % Paraformaldehyde Aqueous Solution (#15710, Electron Microscopy Sciences) to 4 % in PBS
  • Blocking and antibody dilution buffer: 0.3 % Triton X-100, 5 % (v/v) FCS in PBS
  • DAPI staining solution: 300 nM DAPI in PBS (#D1306, ThermoFisher Scientific or similar)
  • Mounting medium: DAKO Fluorescence Mounting medium, # S3023, Agilent or similar
  • Coverslips #1.5 (eg 631-0150, VWR)
  • Slides (eg SuperFrost Plus, J1800AMNZ, Epredia)
  • Primary and secondary antibodies (see table 3 & 4 for antibody suggestions)


Seed cells
Seed cells
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 DurationOvernight .

HEK 293 cell transfection
HEK 293 cell transfection
10m
This protocol uses a DNA: Fugene ratio of 1:3. Prepare the transfection complexes as follows:

ABCD
DNAFugeneSerum-free DMEM
12 well1000 ng3 ul100 ul
24 well500 ng1.5 ul50 ul
Preparation of transfection complexes (quantities are per well)

Add the required amount of plasmid DNA to serum-free DMEM. Mix briefly.
Add the required amount of Fugene HD Transfection Reagent.
Vortex and incubate for Duration00:10:00 at TemperatureRoom temperature .

10m
In the meantime, change the medium on the cells to fresh DMEM containing 10 % FCS
Add Amount50 µL per 24-well or Amount100 µL per 12-well drop-wise to the cells and incubate DurationOvernight

Western Blotting
Western Blotting
25m
This section describes the sample preparation and analysis for Western Blotting.
Wash the cells gentle with PBS
Immediately add Amount100 µL per well of ice-cold cell lysis buffer and place cells on ice.

Scrape cells with a cell scraper and harvest cell lysate into a 1.5 ml Eppendorf tube.
Incubate the cells TemperatureOn ice for Duration00:10:00 , vortex occasionally

10m
Clear the cell lysate by spinning down at Centrifigation16200 x g, 4°C for Duration00:15:00

15m
Transfer the post-nuclear supernatant to a fresh 1.5 ml Eppendorf tube and store at Temperature-20 °C .

Prepare the cell lysates for Western Blotting by adding LDS sample buffer and denaturing agent and denature the samples at Temperature80 °C for Duration00:08:00 .

8m
Run samples on 4-12 % Bis-Tris SDS-page. (approx. Duration00:35:00 at 160 V const.)

35m
Transfer proteins onto a PVDF membrane using the Turbo transfer system (BioRad) or similar.
Block membranes in 5% milk/TBS-T for Duration01:00:00 at TemperatureRoom temperature .

1h
Incubate the membranes with primary antibodies at Temperature4 °C DurationOvernight .
ABCDE
TargetCat #SupplierRaised inDilution
GPNMB (N-terminal)AF2550R&D SystemsGoat1:1000
GFPMA5-15256ThermoFisher ScientificMouse1:1000
ActinA1978SigmaMouse1:5000
Table 1: Primary antibodies for Western Blotting.


Wash the plots in TBS-T for Duration00:05:00 at TemperatureRoom temperature . Repeat this step twice for a total of three washes.

5m
Dilute the secondary antibody in 5% milk/TBS-T and incubate the membranes with secondary antibodies at TemperatureRoom temperature for Duration00:45:00 .
ABC
Antibody suggestionDilution
anti-mouse-Peroxidaseeg, A3682, Sigma1:10000
anti-goat-Peroxidaseeg, A5420, Sigma1:10000
Table 2: Secondary antibodies for Western Blotting.

45m
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.
Immunofluorescence
Immunofluorescence
2h 20m
Gently wash coverslips with PBS and fix in 4 % PFA/PBS for Duration00:15:00 min.

15m
Gently wash the cells with PBS. Replace the PBS and add the permeabilisation/blocking. Incubate coverslips for a minimum of Duration00:20:00 min at TemperatureRoom temperature .

20m
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.
Prepare the antibody staining solution in Blocking and staining buffer. Find a suggestion of antibodies for counterstaining below:
ABCDE
TargetCat #SupplierRaised inDilution
LAMP-1H4A3DSHBMouse1:100
TGN4613573-1-APProteintechRabbit1:100
Table 3: Primary antibodies for immunofluorescence.

Pipette a Amount45 µL drop of the antibody staining solution onto the Parafilm and invert the coverslip onto the staining solution so that the cells face downwards.

Incubate for Duration01:00:00 hr in the dark.

1h
Wash the coverslips three times with PBS.
Prepare a staining solution containing the secondary antibody:
AB
Antibody suggestionDilution
anti-mouse-AF6471:1000
anti-rabbit-AF5861:1000
Table 4: Secondary antibodies for Immunofluorescence.

Pipette a Amount45 µL drop of the antibody staining solution onto the Parafilm and invert the coverslip onto the staining solution so that the cells face downwards.
Incubate for Duration00:45:00 min in the dark.

45m
Wash the coverslips once in PBS, stain with DAPI (or other nuclear stain), and mount onto glass slides.
GPNMB can be observed predominantly at the trans-Golgi network but can also be seen at lysosomal compartments.