Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines

Abstract

Epichaperomes are disease-associated pathologic scaffolds, composed of tightly bound chaperones, co-chaperones, and other factors. They mediate anomalous protein-protein interactions inside cells, which aberrantly affects the function of protein networks, and in turn, cellular phenotypes. Epichaperome study necessitates the implementation of methods that retain these protein complexes in their native cellular states for analysis. Here we describe a protocol for detection and composition analysis of epichaperomes in cell homogenates through native polyacrylamide gel electrophoresis.

Keywords: Chaperones; Epichaperomes; HSP70; Heat shock protein 90 (HSP90); High-order assemblies; Immunoblot; Multimolecular protein complexes; Native polyacrylamide gel electrophoresis (native-PAGE); Non-denaturing gel; Oligomers.

Fig. 1

Electrophoresis system setup. Snapshots of the setup at different stages, as described in the protocol, are provided. a: Cell lysates (100 μg) were loaded onto the gel. Pre-run, for gel equilibration, was done at 100V (cold room). The gel was run at 125V in the cold room. The run time for the 4 to 8 % hand cast gels was 2 h, for the 4 to 10% hand cast gel was 2.5 h, and for the 4 to 20% precast gel (Biorad) was 4 h. Transfer was done with 0.02% SDS in 1× transfer buffer for 2 h (cold room). PVDF membrane was used for the protein transfer process, unless otherwise indicated. b: Setup for Invitrogen 4 to 12% precast gels. The run time for the 4–12% precast gel (Invitrogen) was 3.5 h

Fig. 2

The impact of sample storage and handling on epichaperome stability. Cell lysates (100 μg) were loaded onto the gel (Invitrogen precast 4–12% gradient gel) freshly prepared (1), or after being stored as indicated. For (2) lysates were stored at −20 °C but thawed at least 6 times before use. Pre-run, for gel equilibration, was done at 100V (cold room). The gel was run at 125V in the cold room for 3.5 h. Transfer was done with 0.02% SDS in 1× transfer buffer for 2 h (cold room). PVDF membrane was used for the protein transfer process

Fig. 3

The effect of gel gradient on the separation and detection of epichaperomes in the MDA-MB-468 cancer cell line. Several chaperone and co-chaperone components of epichaperomes were detected as indicated. Setup same as in Fig. 1 for cell lysates (100 μg) loaded onto the Invitrogen precast 4–12% gradient gel or the Biorad 4 to 20% gradient gel

Fig. 4

The influence of gel type on epichaperome separation and detection efficiency. a: Native PAGE profile of epichaperomes in the indicated cancer cell lines. CCD-18 contains little to no epichaperomes and is used as control to show the biochemical signature of chaperones on Native PAGE. Note: the protocol used is same as in Fig. 1, but a roller was used to remove air bubbles, which stretched the gel. Most affected are the 4–12 % precast Invitrogen gels (1mm thickness). The run time for the 4–8 % hand cast gels was 2 h, for the 4 to 10 % hand cast gel was 2.5 h, for the 4–12 % precast gel (Invitrogen) was 3.5 h, and for the 4 to 20% precast gel (Biorad) was 4 h. The hand cast gel was stored at 4 °C for one month prior to use. b: SDS PAGE profile (i.e., total levels) of chaperones in samples from panel (a). Note: additional bands for HSP110 and HSC70 are due to incomplete membrane stripping prior to re-blotting

Fig. 5

Reproducibility by a second investigator. Same as in Fig. 4 for samples run by a different investigator using hand cast gels. A nitrocellulose membrane was used for transfer in this case (for the Native gel only)