Sep 07, 2022
Immunological detection of autophagy and mTORC1-related proteins
- Harper JW1,2,
- Sharan Sharan Swarup3
- 1Department of Cell Biology, Harvard Medical School Boston, MA 02115, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
- 3Harvard Medical School
Protocol Citation: Harper JW, Sharan Sharan Swarup 2022. Immunological detection of autophagy and mTORC1-related proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzr94v8j/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 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68680
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000282
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Abstract
Here we present a general protocol for immunological detection by Western blotting MTOR, MTOR (pS2448), ULK1, ULK1 (pS757), p70S6K, p70S6K (pT389), SQSTM1, CALCOCO2, MAP1LC3B, GABARAP, TFEB, TFE3, PGRN, HSP90, and PCNA
Materials
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Antibodies | |||
| human anti-progranulin | R&D Systems | AF2420 | |
| anti-PCNA | Santa Cruz Biotechnology | sc56 | |
| anti-HSP90 | Proteintech | 60318-1-lg | |
| anti-TFEB | Cell Signaling Technology | 4240 | |
| anti-TFE3 | Proteintech | 14480-1-AP | |
| anti-SQSTM1 | Proteintech | 18420-1-AP | |
| anti-Calcoco2 | Proteintech | 12229-1-AP | |
| anti-MAP1LC3B | Cell Signaling Technologies | 2775 | |
| anti-GABARAP | Proteintech | 18723-1-AP | |
| anti-MTOR | Cell Signaling Technologies | 2983 | |
| anti-p70S6K | Cell Signaling Technologies | 2708 | |
| anti-MTOR (pS2448) | Cell Signaling Technologies | 2971 | |
| anti-ULK1 | Cell Signaling Technologies | 8054 | |
| anti-ULK1 (pS757) | Cell Signaling technologies | 14202 | |
| IRDye 680RD Goat a-Rabbit IgG secondary antibody | Li-Cor | 926-68071 | |
| IRDye 680RD Goat a-Mouse IgG secondary antibody | Li-Cor | 926-68070 | |
| IRDye 800CW Goat a-Rabbit IgG secondary antibody | Li-Cor | 926-32211 | |
| IRDye 800CW Goat a-Mouse IgG secondary antibody | Li-Cor | 926-32210 | |
| Goat a-Rabbit IgG, HRP-linked antibody | Cell Signaling Technology | 7474P2 | |
| Goat a-Rabbit IgG HRP conjugate | Bio-Rad | 1706515 | |
| Goat a-Mouse IgG HRP conjugate | Bio-Rad | 1706516 | |
| Chemicals, peptides, and recombinant proteins | |||
| PhosSTOP | Roche | 04906845001 | |
| Complete EDTA-free protease inhibitor cocktail | Sigma-Aldrich | 11873580001 | |
| REVERT 700 total protein stain kit | Li-Cor | 926-11016 | |
| NuPAGE LDS sample buffer (4X) | Thermo Fisher Scientific | NP0007 | |
| NuPAGE sample reducing agent (10X) | Thermo Fisher Scientific | NP0009 | |
| Bio-Rad Protein Assay Dye Reagent Concentrate | Bio-Rad | 5000006 | |
| NuPAGE MES SDS Running Buffer (20X) | Thermo Fisher Scientific | NP0002 | |
| Immobilon-FL PVDF Membrane | Millipore | IPFL00010 | |
| WHEATON Dounce Tissue Grinder, 7 mL | DWK Life Sciences | 357542 | |
| KIMBLE KONTES Dounce Tissue Grinder, 2 mL | DWK Life Sciences | 885300-0002 | |
| Nonidet P40 substitute | Sigma-Aldrich | 74385 | |
| Urea | Sigma-Aldrich | U5378 | |
| RIPA lysis and extraction buffer | Thermo Fisher Scientific | 89900 | |
| Experimental models: Cell lines | |||
| 293T cells | ATCC | CRL-3216 | |
| 293 cells | ATCC | CRL-1573 | |
| HeLa cells | ATCC | CCL-2 | |
| HeLa cells; GRN-/- | This study and DOI: dx.doi.org/10.17504/protocols.io.4r3l2oxqqv1y/v1 | ||
| Software and algorithms | |||
| ImageLab v6.0.1 | Biorad | https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z&source_wt=imagelabsoftware_surl |
Western blotting
Western blotting
Lyse cell pellets by homogenization in KPBS buffer, urea buffer, or RIPA buffer with protease and phosphatase inhibitors. FOr some experiments, we employ 293 cells or alternatively HeLa cells with or without the GRN gene, created by CRISPR-based gene editing (DOI: dx.doi.org/10.17504/protocols.io.4r3l2oxqqv1y/v1).
Determined total protein concentration by BCA or Bradford assay. Normalize samples within a set of samples with additional buffer. Add NuPAGE LDS buffer (4X) plus NuPAGE reducing agent (10X).
Load samples onto 4-12% NuPAGE Bis-Tris gels (ThermoFisher), and separate by electrophoresis in MES buffer.
Transfer proteins to PVDF or nitrocellulose membranes by standard wet transfer in 20% methanol.
Stain membranes with REVERT 700 total protein stain following manufacturer’s instructions, and image total protein with a ChemiDoc MP (Bio-Rad) at 680 nm.
De-stain with REVERT reversal solution for 5 min. Block membranes with tris-buffered saline (TBS) (5% non-fat dry milk) at room temperature for 30-60 min.
Incubate membranes overnight at 4ºC with primary antibody solution in TBS with 0.1% Tween-20 (TBST). Wash six times with TBST for 5 min each. Incubate in secondary antibody solution in TBST (plus 0.01% SDS) for 1h at room temperature.
Wash membranes four times with TBST for 5 min each.
When using HRP-conjugated secondary antibodies (Bio-Rad or Cell Signaling Technology), apply luminol and hydrogen peroxide solution to membrane for 2 min, and image membrane with a ChemiDoc MP using the chemiluminescent setting.
When using Li-Cor fluorescent secondary antibodies, blot membranes dry and image with a ChemiDoc MP at either 800 nm or 680 nm, depending on the secondary antibody.