Nov 12, 2024
Immunoblotting protocol for dopamine and cortical iPSC-derived neurons with different Parkinsonian mutations
- 1University of Sydney
Protocol Citation: Nicolas Dzamko, Jessica Chedid 2024. Immunoblotting protocol for dopamine and cortical iPSC-derived neurons with different Parkinsonian mutations. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9xpol3p/v1
Manuscript citation:
https://doi.org/10.1101/2024.10.07.617117
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 12, 2024
Last Modified: November 12, 2024
Protocol Integer ID: 111984
Keywords: cortical cells, immunoblot, western blot, PD, alpha synuclein, P62, GBA, Rab-10, B-actin
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol details the method for immunoblotting of differentiated VMDA and cortical iPSC's from different Parkinson's mutations for quantification of Parkin, α-syn, Rab-10 and glucocerebrosidase proteins.
Materials
Solutions
- 1 x MES buffer
50 mL 20x MES Buffer
950 mL dH2O
*make 1 L at a time
- 10 x Transfer buffer (1L):
105 g Glycine
22.5 g Tris
1L dH2O
- 1 x Transfer Buffer (15% methanol)
100 mL 10x Transfer buffer
750 mL dH2O
150 mL Methanol
Note: Should be made up fresh (place in cold room or on ice to cool prior to transfer)
- 10x TBS buffer (1L):
87 g Sodium Chloride
12 g Tris
* pH to 7.4
- 1x TBS + 0.1% Tween 20 (TBS-T)
100 mL 10x TBS buffer
1 mL Tween-20
900 mL dH2O
*use magnetic stirrer to dissolve Tween into solution
- 5% Skim Milk in TBST
5 g skim milk powder
100 mL TBS-T
- Stripping buffer (1L)
15 g glycine
1 g SDS
10 ml Tween20
- Adjust pH to 2.2
- Bring volume up to 1 L with ultrapure water.
Antibodies
| Antibody | Company | Cat. No. | Dil. Factor | Molecular weight | |
| alpha synuclein | BD Biosciences | 610787 | 1:1000 | 14kDa | |
| P62/SQSTM1 | Abcam | ab56416 | 1:1000 | 62 kDa | |
| GBA | Abnova | H00002629-M01 | 1:1000 | 62 kDa | |
| Rab-10 | Cell Signalling Technologies | 8127S | 1:1000 | 23 kDa | |
| B-actin | Abcam | ab6276 | 1:50000 | 37 kDa |
Primary antibodies
| Antibody | Company | Cat. No. | Dil. Factor | |
| Anti-Mouse IgG (H+L) HRP Conjugate | Bio-Rad | 1706516 | 1:5000 | |
| Anti-Rabbit IgG (H+L) HRP Conjugate | Bio-Rad | 1706515 | 1:5000 |
Secondary antibodies
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet) for each of the raw materials used.
Ethics statement
Experiments using human iPSCs were approved by the Human Research Ethics Committee at the
University of Sydney (2017/094). Application of this protocol may need prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Before start
Ventral midbrain progenitors and Cortical neural progenitors were generated from iPSC according to the methods of Gantner and colleagues (1, 2).
BCA Assay and Sample preparation
BCA Assay and Sample preparation
Thaw samples and centrifuge at 12000 rpm, 4°C , 4°C, 00:20:00
Transfer supernatant to new microcentrifuge tubes and discard pellet.
Perform BCA assay as per manufacturer's protocol to determine protein concentration of
lysates
Prepare 30 µL sample for loading by adding 20 ug of protein lysate to required volme of dH2O
and 7.5 µL of 4X NuPage LDS sample Buffer
Store samples at -20 °C until ready to run western blot
Running Gels
Running Gels
1h 10m
1h 10m
Thaw samples from freezer on ice and place on heating block for00:10:00 at 70 °C
10m
While waiting for samples to heat up remove the white sticker and gel comb carefully from
gel(s) and rinse with dH2O.
Place 12-well gel(s) into casket and clip in place. Fill up the centre part with 1 X MES buffer and
check for any leaks. Once in place, add 1 X MES to the outer space until half way full.
After samples have heated, quickly vortex and spin down.
Load 4 µL of the ladder and 25 µL of the samples prepared at 1 mg/mL (or as needed)
Run gel at 180 V (constant voltage) for01:00:00 at Room temperature
1h
Transfer and blocking
Transfer and blocking
2h 30m
2h 30m
Obtain large container and fill with 1 X transfer buffer
For each gel obtain a sandwich apparatus, 2 X sponges, 2 X filter paper, 1 X nitrocellulose
membrane (0.45 um)
Open sandwich apparatus and place components in the following order: sponge, filter paper,
gel, nitrocellulose membrane, filter paper, sponge
Perform transfer at 90 V (constant voltage) for 01:30:00 in cold room
1h 30m
Place membrane in container and cover with 5% skim milk in TBS-T solution, place on rocking
platform for 01:00:00
1h
Primary and secondary antibody incubations
Primary and secondary antibody incubations
1h 20m
1h 20m
Prepare primary antibody in 5% skim milk in TBS-T solution (dilution 1:1000)
Cut blots using scissors into smaller strips as desired
Incubation at 4 °C overnight with gentle rocking
Wash blots with TBS-T every 3 times for 00:10:00
10m
Prepare secondary antibody (anti-mouse or anti-rabbit HRP conjugated antibody) in 2.5% skim
milk in TBS-T solution
Incubation at Room temperature for 01:00:00 with gentle rocking
1h
Wash blots with TBS-T every 3 times for 00:10:00
10m
Blot imaging
Blot imaging
Visualise bands using BioRad ChemiDoc MP Imaging System chemiluminescence mode
Band analysis and quantication is performed in ImageLab, all proteins are normalised to B-
actin used as a loading control
Stripping and
Stripping and
1h 20m
1h 20m
If stripping membrane is required to reprobe withanother antibody, add Abcam mild stripping
buffer incubate for 00:10:00 at Room temperature with rocking.
10m
Wash membrane 3 times with TBS-T for 00:10:00 each, them block in 5% skim milk in
TBS-T solution for 01:00:00
1h 10m
Repeat step 17 onwards with new primary antibody as required.
Protocol references
1. Gantner, C.W., et al., An optimized protocol for the generation of midbrain dopamine neurons under defined conditions. Star Protocols, 2020. 1(2): p.100065.
2. Gantner, C.W., et al., FGF-MAPK signaling regulates human deep-layer corticogenesis. Stem cell reports, 2021. 16(5): p. 1262-1275