Immunoblots
Richard Wade-Martins, Nancy C. Hernandez Villegas, schekman, wusj, Iona Thomas-Wright
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.
Steps
Immunoblots
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. 1h 0m 0s
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 0h 10m 0s and blocking was carried out with 5% skimmed milk.
For immunoblots of endogenous α-syn in SH-SY5Y cells, PVDF membranes were fixed with 0.4% paraformaldehyde in TBST at room temperature for 0h 30m 0s
Blots were washed with TBST for 5 times, each time with 0h 5m 0s agitation.
Incubate membranes with secondary antibodies, anti-rabbit or anti-mouse for 1h 0m 0s at Room temperature
Detection was performed with Supersignal Chemiluminescent substrate and quantified with Fiji/ImageJ.
