Aug 23, 2022
Immunoblotting analysis of samples from GolgiTAG (TMEM115-3HA) immunoprecipitation
- Rotimi Fasimoye1,
- Dario R Alessi1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
- Rotimi Fasimoye: rfasimoye001@dundee.ac.uk
- Dario R Alessi: d.r.alessi@dundee.ac.uk
Protocol Citation: Rotimi Fasimoye, Dario R Alessi 2022. Immunoblotting analysis of samples from GolgiTAG (TMEM115-3HA) immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61oxdvqe/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 16, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68696
Keywords: GolgiTAG, Immunoprecipitation, LRRK2 Signalling Pathway, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000463
Abstract
Analysis of the expression of organelle-specific markers is essential to verify the efficiency of any Golgi immunoprecipitation (IP) protocol. Here, we describe our immunoblotting method to assess efficient enrichment of Golgi proteins in Golgi immunoprecipitation products compared to whole cell lysates, as well as the purity of the immunoprecipitated Golgi by monitoring the expression of other organelles’ markers. This method can also be used to verify the expression of GolgiTAG (TMEM115-3HA) in cells that are transiently or stably transfected.
Note: For a detailed description of our method for Golgi immunoprecipitation, refer to dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1 (Introducing GolgiTag to Cells and Immunoprecipitation of Golgi).
Note: This protocol was adapted from dx.doi.org/10.17504/protocols.io.bsgrnbv6 (Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway).
Attachments
ius3bgrdf.docx
174KB
Materials
Reagents:
Lysis buffer:
| A | B | |
| HEPES-KOH pH 7.5 | 50 mM | |
| Triton X-100 | 1% (v/v) | |
| EGTA | 1 mM | |
| Na3VO4** | 1 mM | |
| NaF | 50 mM | |
| β-glycerophosphate | 10 mM | |
| Sodium pyrophosphate | 5 mM | |
| Sucrose | 0.27 M | |
| cOmpleteTM | ||
| EDTA-free Protease Inhibitor Cocktail (Roche, 11836170001)** | ||
| Microcystin-LR (Enzo Life Sciences, ALX-350-012)** | 1 μg/ml |
**: To be added fresh before use.
cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
Microcystin-LR Enzo Life SciencesCatalog #ALX-350-012
- Pierce™ BCA Protein Assay KitThermo FisherCatalog #23227
- 4X Loading buffer: Invitrogen™ NuPAGE™ LDS Sample Buffer, cat no NP0007;
4X SDS loading buffer:
| A | B | |
| Tris-HCl pH 6.8 | 250 mM | |
| SDS | 8% (w/v) | |
| Glycerol | 40% (v/v) | |
| Bromophenol blue | 0.02% (w/v) |
Note: Supplement with 5% (v/v) beta-mercaptoethanol before use.
- NuPAGE 4–12% Bis–Tris Midi GelThermo Fisher ScientificCatalog #WG1403BOX or a self-cast 10% Bis-Tris gel.
SDS-PAGE buffer:
For NuPAGETM Bis-Tris gels: NuPAGE™ MOPS SDS Running Buffer (20X)Thermo FisherCatalog #NP000102
For self-cast Bis-Tris gels:
| A | B | |
| MOPS | 50 mM | |
| Tris | 50 mM | |
| SDS | 0.1% (w/v) | |
| EDTA | 1 mM |
Protein transfer buffer:
| A | B | |
| Tris-base | 48 mM | |
| Glycine | 39 mM | |
| Methanol (freshly supplemented) | 20% (v/v) |
TBS-T:
| A | B | |
| Tris–HCl pH 7.5 | 50 mM | |
| NaCl | 150 mM | |
| Tween 20 | 0.1% (v/v) |
- Membrane blocking solution: 5% (w/v) non-fat milk powder in TBS-T.
- Antibody dilution buffer: 5% (w/v) bovine serum albumin (BSA) in TBS-T.
- Primary antibodies and near-infrared fluorescent IRDye secondary antibodies (See Table 1 and Table 2).
GM130 (D6B1) XP® Rabbit mAbCell Signaling TechnologyCatalog #12480
Golgin-97 (D8P2K) Rabbit mAbCell Signaling TechnologyCatalog #13192
HASigmaCatalog #11867423001
HSP60 (D6F1) XP® Rabbit mAbCell Signaling TechnologyCatalog #12165
VDACCatalog #4661
Calreticulin (D3E6) XP® Rabbit mAbCell Signaling TechnologyCatalog #12238
LAMP1 (C54H11) Rabbit mAbCell Signaling TechnologyCatalog #3243
Alpha-tubulinCell Signaling TechnologyCatalog #3873S
Table 1:
| A | B | C | D | E | |
| Antibody Target | Company | Cat.Number (RRID) | Host species | Dilution | |
| GM130 (Golgi marker) | Cell Signalling Technology | 12480 (RRID:AB_2797933) | Rabbit | 1:1000 | |
| GOLGIN97 (Golgi marker) | Cell Signalling Technology | 13192 (RRID:AB_2798144) | Rabbit | 1:500 | |
| ACBD3 (Golgi marker) | Sigma Atlas | HPA015594 (RRID:AB_1844491) | Rabbit | 1:1000 | |
| HA | Sigma | 11867423001 (RRID:AB_390918) | Rat | 1:1000 | |
| HSP60 (mitochondrial marker) | Cell Signalling Technology | 12165 (RRID:AB_2636980) | Rabbit | 1:1000 | |
| VDAC (mitochondrial marker) | Cell Signalling Technology | 4661 (RRID:AB_10557420) | Rabbit | 1:1000 | |
| Calreticulin (ER marker) | Cell Signalling Technology | 12238 (RRID:AB_2688013) | Rabbit | 1:1000 | |
| LAMP1 (lysosomal marker) | Cell Signalling Technology | 3243 (RRID:AB_2134478) | Rabbit | 1:1000 | |
| CSTC (lysosomal marker) | Santa Cruz | Sc-74590 (RRID:AB_2086955) | Mouse | 1:1000 | |
| alpha-tubulin (cytoplasmic marker) | Cell Signalling Technology | 3873S (RRID:AB_1904178) | Mouse | 1:5,000 |
IRDye® 800CW Donkey anti-Mouse IgG Secondary AntibodyLicorCatalog #926-32212
IRDye® 800CW Donkey anti-Rabbit IgG Secondary AntibodyLicorCatalog #926-32213
Goat anti-Rat IRDye 800CWLI-CORCatalog #926-32219
Table 2:
| A | B | C | |
| Secondary Antibodies | Company | Cat. Number (RRID) | |
| Donkey anti-mouse IRDye 800CW | LI-COR | 926-32212 (RRID:AB_621847) | |
| Donkey anti-rabbit IRDye 800CW | LI-COR | 926-32213 (RRID:AB_621848) | |
| Goat anti-Rat IRDye 800CW | LI-COR | 926-32219 (RRID:AB_1850025) |
Equipment:
- Refrigerated bench-top centrifuge (Eppendorf microcentrifuge 5417R, or equivalent).
- Plate reader for Protein quantification (BioTek Epoch, or equivalent).
- Digital Dry Bath/Block Heater, 2 block configuration, CN, EU, UK plugs, 200-240VThermo FisherCatalog #88870005
- SureLock Midi-Cell Electrophoresis System (if using Invitrogen NuPAGE precast midi gels), or equivalent gel electrophoresis apparatus.
- Protein transfer apparatus: Trans-Blot® Cell (Bio-Rad), or equivalent wet transfer system.
- See-saw rocker (VWR SSL4, or equivalent).
- Odyssey CLx Imaging System paired with Image StudioTM Software.
Preparation of IP samples or whole cell lysates samples:
Preparation of IP samples or whole cell lysates samples:
For a detailed description of our method for Golgi immunoprecipitation, refer to dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1 (Introducing GolgiTag to Cells and Immunoprecipitation of Golgi). This also includes a description of how to prepare whole cell lysates samples.
Preparation of samples for immunoblot analysis:
Preparation of samples for immunoblot analysis:
Determine the protein concentration of IP or cell lysate samples by BCA Protein Assay Kit according to the manufacturer’s instructions, performing measurements in triplicate.
Note
Note: Ensure the concentration of the samples is in the linear range for the BCA Protein Assay. If it is not, prepare appropriate dilutions in water of each lysate. Generally, protein concentrations of IP samples should range from 0.1 µg/µL to 0.3 µg/µL . For near confluent cells lysed as described above, protein concentration should range from 0.5 µg/µL to 5 µg/µL (depending on cell type).
Prepare samples for immunoblotting to achieve the same protein concentration for all samples (ideally, 0.1 µg/µL -2 µg/µL , depending on the sample at the lowest concentration).
Dilute with lysis buffer as appropriate.
Add a quarter of a volume of 4X SDS/LDS loading buffer freshly supplemented with beta-mercaptoethanol (i.e. for 7.5 µL of lysate/lysis buffer mix, add 2.5 µL of loading buffer).
Mix by vortexing.
Incubate samples for 00:05:00 at 70 °C heating block before immunoblot analysis.
Note
Note: if blotting for TMEM115, do not heat sample. We noticed that boiling leads to the TMEM115 not going into the resolving gel during electrophoresis.
5m
SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
Load samples (2 µg for IP samples or 10 µg for whole cell lysate samples) onto a NuPAGE 4–12% Bis–Tris Midi Gel (ThermoFisherScientific, Cat#WG1402BOX or Cat#WG1403BOX), or a self-cast 10% Bis-Tris gel, alongside pre-stained molecular weight markers (ranging from 10 kDa to 250 kDa). Rinse wells carefully with running buffer before loading samples.
Note
- For optimal signal, the amount of protein loaded for IP samples is 2 µg while for whole cell lysate sample ranges from 2 µg -20 µg .
- Be aware of maximum loading capacity of each well as per manufacturer’s instructions and take care not to overload wells.
- If multiple gels are used for each set of experimental samples, an internal loading control should lso be included for subsequent data normalization.
Electrophorese samples at 120V with MOPS SDS running buffer for 02:00:00 or until the blue dye runs off the gel.
2h
Protein transfer (Wet electroblotting):
Protein transfer (Wet electroblotting):
Equilibrate the gel, one piece of nitrocellulose membrane (GE Healthcare, Amersham Protran Supported 0.45 µm NC) and two pieces of filter paper (WhatmanTM3MM Chr Chromatography Paper, or equivalent) (all the same size as the gel) by pre-soaking them in transfer buffer.
Assemble the gel and membrane transfer stack in a tray filled with transfer buffer to ensure that all components are submerged during the assembling.
Place one sponge pad inside the cassette holder (on the side that will be facing the cathode).
Place one piece of filter paper on top of the sponge pad, followed by the gel, nitrocellulose membrane, another piece of filter paper and another sponge pad.
Note
Note: Carefully remove any air bubbles between layers using a roller after adding each layer.
Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.
Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 90 V (constant voltage) for 01:30:00 On ice using a wet transfer system.
1h 30m
After transfer, stain membranes with Ponceau solution to assess transfer efficiency and general quality of the samples.
Note
If an image is required for record, the Ponceau-stained membraned can be scanned.
Each membrane can be divided into two sections by one horizontal cut (just above the 50 kDa ladder band): ‘top section’ (from the top of the membrane to the 50 kDa marker) and ‘bottom section’ (from the 50 kDa marker to the bottom of the membrane) (Figure 1), to be incubated with primary antibodies as described in step 14.
Note
Notes: When immunoblotting using anti-Tubulin, it is preferable not to cut the membrane as the protein is 50 kDa.
Membrane blocking and antibody incubation:
Membrane blocking and antibody incubation:
Destain membranes from Step 2 by washing with TBS-T and incubate in blocking solution for at least 00:15:00 at Room temperature on a see-saw rocker.
15m
Rinse the membrane in TBS-T and incubate Overnight at 4 °C with primary antibodies (diluted in 5% (w/v) BSA in TBS-T to their working concentration – Table 1), as follows:
Note
Notes: Table 1 lists the primary antibodies (and suggested working dilution) mostly used in our lab to study the GolgiTAG and Golgi proteins and to check for contamination from other organelles. The selectivity and specificity of the antibodies suggested in Table 1 have been extensively validated using appropriate controls. All antibodies listed in Table 1 react with samples from human cells.
After incubation with primary antibodies, wash membranes in TBS-T (3 washes, 5-10 minutes each, on a see-saw rocker).
Wash membranes in TBS-T for 00:05:00 -00:10:00 on a see-saw rocker(1/3).
15m
Wash membranes in TBS-T for 00:05:00 -00:10:00 on a see-saw rocker(2/3).
15m
Wash membranes in TBS-T for 00:05:00 -00:10:00 on a see-saw rocker(3/3).
15m
Incubate membranes with near-infrared fluorescent dye-labelled secondary antibodies (diluted to the working concentration: 1:20,000) for 01:00:00 at Room temperature on a see-saw rocker.
Note
Note: Table 2 lists the near-infrared fluorescent dye-labelled secondary antibodies used in our lab.
1h
Extensively wash membranes in TBS-T (4 washes, 00:10:00 -00:15:00 each, with agitation).
25m
Wash membranes in TBS-T for 00:10:00 -00:15:00 with agitation(1/4).
25m
Wash membranes in TBS-T for 00:10:00 -00:15:00 with agitation(2/4).
25m
Wash membranes in TBS-T for 00:10:00 -00:15:00 with agitation(3/4).
25m
Wash membranes in TBS-T for 00:10:00 -00:15:00 with agitation(4/4).
25m
Image acquisition and Analysis:
Image acquisition and Analysis:
Acquire the protein bands via near infrared fluorescent detection using the Odyssey CLx Imaging System and the signal intensity quantified using the Image Studio Software.
Note
Note: To control for inter-gel variability, the signal intensity of each band can be normalised against the control sample loaded in each gel of a set of experiments.
Analyse immunoblotting data using a software for statistical analysis (GraphPad Prism, or equivalent).
Figure 1: Representation of how to divide Ponceau-stained membranes into two separate halves for subsequent incubation with primary antibodies. Each membrane is cut into two halves just above the 50kDa marker (along the dotted line). The top section is incubated with primary antibodies against GM130, GOLGIN97, ACBD3, HSP60, LAMP1 and Calreticulin while the bottom section is incubated with primary antibodies against HA (could also be incubated with VDAC and CTSC).
Figure 2: Representative results of immunoblotting analysis of samples from whole cell lysate and Golgi IP. Left panel shows the detection of C-terminal HA-tagged TMEM115 using an anti-HA antibody. Right panel shows the detection of different organelle markers from whole cell lysates and IP samples. Samples were prepared according to the protocol described in doi…. (Introducing GolgiTag to Cells and Immunoprecipitation of Golgi).). WCL: whole cell lysate; IP: Golgi immunoprecipitation samples; ER: endoplasmic reticulum; Mito: mitochondria; Lyso: lysosome; Cyto: cytoplasm. Note: VDAC and CTSC blots are not shown.