TITLE: Immunoblotting analysis of samples from GolgiTAG (TMEM115-3HA) immunoprecipitation
AUTHORS: Rotimi Fasimoye, Dario R Alessi

[DESCRIPTION]
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).

[STEPS]
SECTION: Preparation of IP samples or whole cell lysates samples:
1. 
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.

SECTION: Preparation of samples for immunoblot analysis:
2. 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.
 

SECTION: Preparation of samples for immunoblot analysis:
3. 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).
SECTION: Preparation of samples for immunoblot analysis:
4. Incubate samples for 5 min at 70 °C heating block before immunoblot analysis.
 


SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
5. 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.
 

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
6. Electrophorese samples at 120V with MOPS SDS running buffer for 120 min or until the blue dye runs off the gel.
SECTION: Protein transfer (Wet electroblotting):
7. Equilibrate the gel, one piece of nitrocellulose membrane (GE Healthcare, Amersham Protran Supported   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.
SECTION: Protein transfer (Wet electroblotting):
8. Assemble the gel and membrane transfer stack in a tray filled with transfer buffer to ensure that all components are submerged during the assembling.
SECTION: Protein transfer (Wet electroblotting):
9. Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.
SECTION: Protein transfer (Wet electroblotting):
10. Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 90 V (constant voltage) for 90 min on ice using a wet transfer system.

SECTION: Protein transfer (Wet electroblotting):
11. After transfer, stain membranes with Ponceau solution to assess transfer efficiency and general quality of the samples.
 

SECTION: Protein transfer (Wet electroblotting):
12. 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.
 

SECTION: Membrane blocking and antibody incubation:
13. Destain membranes from Step 2 by washing with TBS-T and incubate in blocking solution for at least 15 min at Room temperature on a see-saw rocker.
SECTION: Membrane blocking and antibody incubation:
14. Rinse the membrane in TBS-T and incubate   at 4 °C with primary antibodies (diluted in 5% (w/v) BSA in TBS-T to their working concentration – Table 1), as follows:
 


SECTION: Membrane blocking and antibody incubation:
15. After incubation with primary antibodies, wash membranes in TBS-T (3 washes, 5-10 minutes each, on a see-saw rocker).
SECTION: Membrane blocking and antibody incubation:
16. Incubate membranes with near-infrared fluorescent dye-labelled secondary antibodies (diluted to the working concentration: 1:20,000) for 60 min at Room temperature on a see-saw rocker.
 

SECTION: Membrane blocking and antibody incubation:
17. Extensively wash membranes in TBS-T (4 washes, 10 min -15 min each, with agitation).
SECTION: Image acquisition and Analysis:
18. 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.
 

SECTION: Image acquisition and Analysis:
19. Analyse immunoblotting data using a software for statistical analysis (GraphPad Prism, or equivalent).
  
 
SECTION: Preparation of samples for immunoblot analysis:
3.1.  Dilute with lysis buffer as appropriate.
SECTION: Preparation of samples for immunoblot analysis:
3.2.  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).
SECTION: Preparation of samples for immunoblot analysis:
3.3. Mix by vortexing.
SECTION: Protein transfer (Wet electroblotting):
8.1. Place one sponge pad inside the cassette holder (on the side that will be facing the cathode).
SECTION: Protein transfer (Wet electroblotting):
8.2. 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.
 

SECTION: Membrane blocking and antibody incubation:
15.1. Wash membranes in TBS-T for 5 min -10 min on a see-saw rocker(1/3).
SECTION: Membrane blocking and antibody incubation:
15.3. Wash membranes in TBS-T for 5 min -10 min on a see-saw rocker(3/3).
SECTION: Membrane blocking and antibody incubation:
15.2. Wash membranes in TBS-T for 5 min -10 min on a see-saw rocker(2/3).
SECTION: Membrane blocking and antibody incubation:
17.1. Wash membranes in TBS-T for 10 min-15 min with agitation(1/4).
SECTION: Membrane blocking and antibody incubation:
17.2. Wash membranes in TBS-T for 10 min-15 min with agitation(2/4).
SECTION: Membrane blocking and antibody incubation:
17.4. Wash membranes in TBS-T for 10 min-15 min with agitation(4/4).
SECTION: Membrane blocking and antibody incubation:
17.3. Wash membranes in TBS-T for 10 min-15 min with agitation(3/4).