Apr 01, 2022
Version 2
Immunological detection of APP and proteins of the endolysosomal system V.2
- Hankum Park1,2,3,
- Frances V Hundley1,2,
- Harper JW1,2
- 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;
- 3Current affiliation: Seoul National University, School of Dentistry
Protocol Citation: Hankum Park, Frances V Hundley, Harper JW 2022. Immunological detection of APP and proteins of the endolysosomal system . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg36jeeg25/v2Version created by Frances V Hundley
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: March 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59924
Keywords: ASAPCRN
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Abstract
Here we present a general protocol for immunological detection by Western blotting of APP and proteins of the endolysosomal system, including EEA1, RAB5, PSEN1, LAMP1, LAMP2, TMEM192, and BACE1.
Materials
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Antibodies | |||
| a-EEA1 (C45B10) rabbit mAb | Cell Signaling Technology | 3288 | |
| a-RAB5 (C8B1) rabbit mAb | Cell Signaling Technology | 3547 | |
| a-PSEN1 (D39D1) rabbit mAb | Cell Signaling Technology | 5643 | |
| a-PSEN2/AD5 (EP1515Y) rabbit mAb | Abcam | ab51249 | |
| a-LAMP1 (D2D11) rabbit mAb | Cell Signaling Technology | 9091 | |
| a-LAMP2 (D5C2P) rabbit mAb | Cell Signaling Technology | 49067 | |
| a-TMEM192 rabbit pAb | Proteintech | 28263-1-AP | |
| a-HA | Biolegend | 901513 | |
| a-HA (6E2) mouse mAb | Cell Signaling Technology | 2367 | |
| a-FLAG M2 mouse mAb | Sigma-Aldrich | F1804 | |
| a-ZO-1 rabbit pAb | Proteintech | 21773-1-AP | |
| a-Golga1 rabbit pAb | Proteintech | 12640-1-AP | |
| a-Calreticulin rabbit pAb | Proteintech | 10292-1-AP | |
| a-S6K rabbit pAb | Proteintech | 14485-1-AP | |
| a-RAB11 (D4F5) rabbit mAb | Cell Signaling Technology | 5589 | |
| a-Lamin A/C (4C11) mouse mAb | Cell Signaling Technology | 4777 | |
| a-VDAC1/Porin rabbit pAb | Proteintech | 55259-1-AP | |
| a-RAB7 (D95F2) rabbit mAb | Proteintech | 9367 | |
| a-DYKDDDDK tag, mouse mAb (FG4R) | Thermo Fisher Scientific | MA1-91878 | |
| a-GAPDH (D16H11) XP rabbit mAb | Cell Signaling Technology | 5174 | |
| a-APP CTF (C1/6.1) mouse mAb | BioLegend | 802801 | |
| a-APP A4 (22C11) mouse mAb | Sigma | MAB348 | |
| a-PEX19 rabbit pAb | Proteintech | 14713-1-AP | |
| a-CD71/TFR1 (D7G9X) rabbit mAb | Cell Signaling Technology | 13113 | |
| a-HSP90 (3F11C1) mouse mAb | Proteintech | 60318-1-Ig | |
| a-BACE1 (D10E5) rabbit mAb | Cell Signaling Technology | 5606 | |
| 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 | |
| 293L: TMEM192-3xHA | This study | ||
| 293L-APP-/-: TMEM192-3xHA; APP-/- | This study | ||
| 293EL-APP-/-: TMEM192-3xHA; APP-/-; FLAG-EEA1 | This study | ||
| 293EL-APP*: TMEM192-3xHA; APP-/-; FLAG-EEA1; APPSw;T700N | This study | ||
| 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 293EL APPSw,T700N cells expressing 3xFLAG-EEA1, TMEM192-3xHA, and APP harboring Swedish and T700N mutations as described in dx.doi.org/10.17504/protocols.io.byi7puhn.
Determined total protein concentration by BCA or Bradford assay. Normalize samples within a set of samples with additional lysis 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) plus 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) and 5% non-fat dry milk. Wash six times with TBST for 5 min each. Incubate in secondary antibody solution in TBST (plus 0.01% SDS and 5% non-fat dry milk) 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.