Aug 26, 2023
Immunoblots
- wusj1,
- Nancy C. Hernandez Villegas2,
- Iona Thomas-Wright3,
- Richard Wade-Martins3,
- schekman1
- 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States;
- 2Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States;
- 3Oxford Parkinson’s Disease Centre, Department of Physiology, Anatomy and Genetics and Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, United Kingdom
Protocol Citation: wusj, Nancy C. Hernandez Villegas, Iona Thomas-Wright, Richard Wade-Martins, schekman 2023. Immunoblots. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj388wlk5/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: August 04, 2023
Last Modified: August 26, 2023
Protocol Integer ID: 85963
Funders Acknowledgement:
Nancy C Hernandez Villegas
Grant ID: NIH training program T32GM139780
Randy Schekman
Grant ID: Howard Hughes Medical Institute
Richard Wade-Martins
Grant ID: ASAP-020370
Abstract
This protocol describes a standard procedure for protein separation and identification of protein of interest.
Protocol overview:
The steps of this protocol is similar for different sources of protein such as cell lysate, cytosol, or membrane samples, and EV samples. However, there are some specific changes depending on protein of interest, endogenous alpha-synuclein, or type of cells, hiPSC dopamine neurons.
Materials
Reagents
| Reagent | Catalog number | Manufacturer | |
| Gel, 4-20%, 15 wells, WedgeWell | XP04205BOX | Thermo Fisher Scientific | |
| Gel, 4-20% Criterion, 26 wells | 345-0034 | BioRad | |
| Gel, 4-20%, 10 wells, WedgeWell | XP04200BOX | Thermo Fisher Scientific | |
| PVDF, Immobilon P | IPVH00010 | EMD Millipore | |
| Tris base | BP152-5 | Fisher Scientific | |
| Glycine | BP381-5 | Fisher Scientific | |
| Methanol | |||
| PageRuler prestained protein ladder | 26617 | Thermo Scientific | |
| TBS | |||
| Triton X100 | T8787-250ML | Sigma-Aldrich | |
| BSA | A3294 | Sigma |
Antibodies
| Antibodies | Catalog number | Manufacturer | Dilutions | |
| Mouse monoclonal anti-α-synuclein | Cat# 610787 | BD Biosciences | 1:500 | |
| Rabbit polyclonal anti-α-synuclein | Cat# 10842-1-AP | Proteintech | 1:500 | |
| Rabbit polyclonal anti-DNAJC5 | Cat# 144-10489-200 | RayBiotech | 1:1,000 | |
| Mouse monoclonal anti-alpha tubulin | Cat# ab7291 | Abcam | 1:2,000 | |
| Mouse monoclonal anti-Alix | Cat# Sc-53540 | Santa Cruz Biotechnology | 1:1,000 | |
| Rabbit monoclonal anti-CD9 | Cat# 13174S | Cell Signaling Technology | 1:1,000 | |
| Mouse monoclonal anti-PDI | Cat# ADI-SPA-891-D | Enzo Life Sciences | 1:1,000 | |
| Mouse monoclonal anti-CD63 | Cat# BDB556019 | Thermo Fisher Scientific | 1:1,000 | |
| Mouse monoclonal anti-Flotillin-2 | Cat# 610383 | BD Biosciences | 1:1,000 | |
| Mouse monoclonal anti-Transferrin Receptor | Cat# 13-6800 | Thermo Fisher Scientific | 1:1,000 | |
| Mouse monoclonal anti-GM130 | Cat# 610823 | BD Biosciences | 1:1,000 | |
| Rabbit monoclonal anti-Tom20 | Cat# 42406S | Cell Signaling Technology | 1:1,000 | |
| Rabbit polyclonal anti-GFP | Cat# NC9589665 | Fisher Scientific | 1:1,000 | |
| Rabbit polyclonal anti-LC3B | Cat# NB100-2220 | Novus Biologicals | 1:1,000 | |
| Rabbit monoclonal anti-Citrate Synthase | Cat# 14309S | Cell Signaling Technology | 1:1,000 | |
| Rabbit polyclonal anti-Dopamine transporter | Cat# BS-1714R | Bioss Antibodies | 1:1,000 | |
| Rabbit monoclonal anti-beta III Tubulin | Cat# ab215037 | Abcam | 1:1,000 | |
| Rabbit polyclonal anti-Tyrosine hydroxylase | Cat# AB152 | Millipore | 1:1,000 | |
| Chicken polyclonal Microtubule-associated protein 2 | Cat# ab92434 | Abcam | 1:1,000 | |
| Mouse monoclonal anti-FLAG | Cat# F9291 | Sigma-Aldrich | 1:1,000 |
Immunoblots
Immunoblots
40m
40m
Cell lysate, cytosol, or membrane samples, and EV samples were mixed with SDS sample loading
buffer. For cell lysate, cytosol or membrane samples, 20 μg proteins were loaded. For EV samples, the maximal amount up to 20uL were loaded.
Samples with DNAJC5 and α-syn were heated at 55°C for 10 min to prevent aggregation. Other samples were heated at 95°C for 5 min and separated on SDS-PAGE gels (Novex wedgewell 4%-20% Tris-Glycine mini gels, 200 V for 45 min).
Proteins were transferred to PVDF membranes (EMD Millipore, Darmstadt, Germany) in cold room at 0.6 A for 2 h.
Blocked membrane with 5% bovine serum albumin in TBST (20 mM Tris pH 7.4, 150 mM NaCl, and 0.1% Tween-20) at Room temperature for 1 h with constant agitation. 01:00:00
1h
Incubated membranes with primary antibodies at 4 °C overnight.
For immunoblots from hiPSC dopamine neurons, samples in loading buffer were heated to 70 °C for 00:10:00 and blocking was carried out with 5% skimmed milk.
10m
For immunoblots of endogenous α-syn in SH-SY5Y cells, PVDF membranes were fixed with 0.4% paraformaldehyde in TBST at room temperature for 00:30:00
30m
Blots were washed with TBST for 5 times, each time with 00:05:00 agitation.
5m
Incubate membranes with secondary antibodies, anti-rabbit or anti-mouse for 01:00:00 at Room temperature
1h
Detection was performed with Supersignal Chemiluminescent substrate and quantified with Fiji/ImageJ.