TITLE: Cell lysis and gel electrophoresis for protein analysis of HeLa cells
AUTHORS: OLIVIA HARDING, Erika L.F. Holzbaur

[DESCRIPTION]
Here, we present multiple protocols used for biochemical analysis of protein expression and association. First, we used a simple lysis technique to determine the efficiency of an siRNA knockdown. Then, we modified two previously published methods for assaying co-precipitation of p62 and NEMO with magnetic beads conjugated to a GFP-trap molecule. In the first, we pulled down EGFP-NEMO in control or mitochondrial damaged conditions, and in the second, we pulled down EGFP-Ubiquitin in p62-/- cells with expression of wild-type p62 or a dysfunctional mutant. Since p62 is known to form multimers, we used specialized buffers to preserve those putative interactions. We were able to reproduce results published previously by pulling down EGFP-Ubiquitin in p62-expressing cells. However, interestingly, we did not find evidence that NEMO interacts with p62 in the soluble fraction, or via ubiquitin chains generated in basal conditions. These studies demonstrated that NEMO recruitment to damaged mitochondria occurs in specific circumstances, and NEMO colocalization with p62 is also dependent on multiple factors. 

[BEFORE_START]

The start point for this protocol is after cells grown on 3.5 cm, or 10cm dishes have been transfected with relevant constructs for 1080 min- 1440 min and treated with appropriate small molecules or vehicles.
For 3.5 cm dishes, follow transfection procedures enumerated in imaging protocols.
Chill all reagents on ice.
Add protease and phosphatase inhibitors to 1X to each lysis buffer immediately before use.




[GUIDELINES]

 This protocol was developed to analyze protein expression and enrichment in cell culture, including HeLa-M cells and HeLa p62-/- cells.
Option 2 was modified from a protocol used in Turco et al, Molec. Cell, 2019.
Option 3 was modificed from a protocol used in Wurzer et al, eLife, 2015.

[STEPS]
SECTION: Wash cells
1. Aspirate media from dishes.
SECTION: Wash cells
2. Wash samples quickly x2 with ice cold PBS.
 

SECTION: Wash cells
3. 
 
 
Add 150 µL RIPA + inhibitors to dish and scrape cells into 1.5 uL tube, OR add buffer to thawed sample and resuspend by pipetting.
 
SECTION: Wash cells
4. Rotate resuspended sample on end-over-end machine at 4 °C for 20 min.

SECTION: Wash cells
5. Spin at top speed (17000 x g), 4 °C, 20 min.

SECTION: Wash cells
6. Remove supernatant as Lysis and keep on ice or store at -80 °C.

SECTION: Wash cells
7. Measure protein concentration with Pierce BCA assay by adding 25 µL sample or BSA standard to each well in duplicate and 200 µL Reagent A+B. Incubate 37 °C for 30 min then measure absorbance on a plate reader.
 

SECTION: Wash cells
8. Add 1/3 volume of 4X DB to remaining Lysis or a measured fraction of sample and heat 95 °C for 5 min.

SECTION: Wash cells
9. Proceed to gel electrophoresis.
SECTION: Wash cells
3. The samples for this assay are: A) EGFP-vector + veh, B) EGFPvector + AntA/OligA, C) EGFP-NEMO + veh, and D) EGFP-NEMO + AntA/OligA. Start with 3x 10 cm dish per condition for one biological replicate.
 
SECTION: Wash cells
4. 18- 24 hr before collection, transfect 3 µg Parkin and 1.5 µg EGFPNEMO (or vector), to 70-80% confluent cells on each dish.

SECTION: Wash cells
5. It is best to plan collection times such that each is collected and snap frozen ~10 min apart. Then all snap frozen samples can be processed simultaneously from that step.

SECTION: Lysis with heavy fraction isolation
6.  Scrape and combine like samples into 800 µL PBS, transfer to 1.5mL tube.
 
SECTION: Lysis with heavy fraction isolation
7. Spin 5 min, 2000 x g, at 4 °C.

SECTION: Lysis with heavy fraction isolation
8. Aspirate PBS and snap freeze in liquid nitrogen.
SECTION: Lysis with heavy fraction isolation
9.  Add 800 µL LB-A with inhibitors to each frozen sample.

SECTION: Lysis with heavy fraction isolation
10. Pipet to resuspend.
SECTION: Lysis with heavy fraction isolation
11. Incubate on ice 10 min.

SECTION: Lysis with heavy fraction isolation
12.  Centrifuge at 1000 x g, 5 min, 4 °C.

SECTION: Lysis with heavy fraction isolation
13.  Transfer sup. (Cytosol) to a new tube.
SECTION: Lysis with heavy fraction isolation
14.  Centrifuge again at top speed, 10 min, 4 °C.

SECTION: Lysis with heavy fraction isolation
15. Save pellet as Heavy Fraction if desired and resuspend in 50 µL 1X DB.

SECTION: Prep IP beads
16. 
Mix GFP-Trap beads by swirling and gently pipetting to break up accumulations.
SECTION: Prep IP beads
17. Aliquot 25 µL bead slurry into LoBind tubes (one for each condition).

SECTION: Prep IP beads
18. Remove supernatant from beads by inserting the tube in a magnetic rack and carefully aspirating clear solution.
SECTION: Prep IP beads
19. Resuspend beads in 500 µL cold PBST.

SECTION: Prep IP beads
20.  Insert to magnetic rack and remove PBST wash.
SECTION: Prep IP beads
21. Block beads in 2% BSA (in water) for 60 min at 4 °C with end-over-end rotation.

SECTION: Prep IP beads
22.  Wash 2X with 500 µL  Room temperature PBST.

SECTION: Prep IP beads
23.  Wash 1X with 500 µL LB-A.

SECTION: Prep IP beads
24.  Allow magnets to collect beads for up to 5 min.

SECTION: Bead capture
25. 
Add 500 µL Cytosol to bead tube, rotate end-over-end gently for 60 min, 4 °C (save the rest of Cytosol for Input).
 
SECTION: Bead capture
26. Remove supernatant by magnet (up to 5 min) and save as Flow Through.

SECTION: Bead capture
27. Wash 3X with 500 µL  Room temperature. PBS (no TWEEN) (can save these washes).

SECTION: Bead capture
28.  For the last wash, transfer beads and wash to a new tube.
 

SECTION: Bead capture
29. Remove sup from last wash and resuspend in ~90 µL 1X DB with rough mixing (vortex, pipetting), then heat 95 °C for 5 min.

SECTION: Bead capture
30. Magnetize 5 min and save sup as Eluate.

SECTION: Bead capture
31. Add 1/3 volume of 4X DB to Input and Flow Through and heat 95 °C for 5 min.

SECTION: Bead capture
32. Proceed to gel electrophoresis.
SECTION: Wash cells
3. The samples for this assay are in p62-/- Hela cells: A) EGFP-vector + mCherry-vector, B) EGFP-vector + mCherry-p62, C) EGFPUbiquitin + mCherry-vector, D) EGFP-Ubiqutin + mCherry-p62, and E) EGFP-Ubiquitin + mCherry-p62-ΔPB1. Start with 2x 10 cm dish per condition for one biological replicate.
 
SECTION: Wash cells
4. 18- 24 hr before collection, transfect 3 µg Parkin and EGFPUbiquitin (or vector), 1.5 µg NEMO, and 2 µg - 2.5 µg mCherry-p62 (or vector) to 70-80% confluent cells in each dish.
 

SECTION: Lysis
5. 
Combine samples by scraping cells into 600 µL LB-B with inhibitors and transfer to 1.5 mL tube.
 
SECTION: Lysis
6. Incubate on ice, 15 min.

SECTION: Lysis
7.  Centrifuge at 16100 x g (16.1 G), 13 min, 4 °C.

SECTION: Lysis
8.  Transfer supernatant to a new tube as Lysis.
SECTION: Prep IP beads
9.  
Mix GFP-Trap beads by swirling or gently pipetting to break up accumulations and aliquot 10 µL  into LoBind tubes (one for each condition).
 
SECTION: Prep IP beads
10. Remove supernatant from beads by inserting the tube in a magnetic rack and carefully aspirating clear solution.
SECTION: Prep IP beads
11.  Resuspend beads in 500 µL  Room temperature Wash Buffer.

SECTION: Prep IP beads
12.  Insert to magnetic rack and remove wash.
SECTION: Prep IP beads
13. Repeat for a total of 2 washes.
SECTION: Prep IP beads
14.  Wash 1X with 500 µL LB-B.

SECTION: Prep IP beads
15. Allow magnets to collect beads for up to 5 min.

SECTION: Bead capture
16. 
Add 500 µL Lysed sample to bead tube, rotate gently for 60 min, Room temperature.
 
SECTION: Bead capture
17. Save the rest of the Lysis as Input.
SECTION: Bead capture
18. Remove supernatant by magnet (up to 5 min) and save as Flow Through.

SECTION: Bead capture
19. Wash 3X with 500 µL Room temperature Wash buffer (can save these washes).

SECTION: Bead capture
20. For the last wash, transfer beads and wash to a new tube (optional if you’re using Lo-bind tubes).
SECTION: Bead capture
21. Remove supernatant from last wash and resuspend in ~90 µL 1X DB with rough mixing (vortex, pipetting), then heat block 10 min.

SECTION: Bead capture
22.  Magnetize 5 min to collect beads and save supernatant as Eluate.

SECTION: Bead capture
23. Add 1/3 volume of 4X DB to Input and Flow Through and heat 95 °C for 5 min.

SECTION: Bead capture
24. Proceed to gel electrophoresis.
SECTION: Gel electrophoresis and immune-blotting
25. 
 

SECTION: Gel electrophoresis and immune-blotting: Set-up
26. Set up electrophoresis cell with 10% gels by manufacturer’s instructions.
SECTION: Gel electrophoresis and immune-blotting: Set-up
27. Fill cell with RB and flush wells with a plastic transfer pipet.
SECTION: Gel electrophoresis and immune-blotting: Set-up
28. Invert samples by hand to mix, and ensure all samples are at the bottom of tubes by briefly centrifuging.
SECTION: Gel electrophoresis and immune-blotting: Set-up
29. Load wells with equal amounts of protein (Option 1) or equal volumes (Options 2 and 3) and molecular weight standard (4 µL- 5 µL).
 



SECTION: Gel electrophoresis and immune-blotting: Set-up
30. For empty lanes, load approx. equal volume of 1X DB.
SECTION: Gel electrophoresis and immune-blotting: Running
31. Run samples through stacking gel (85 V, 20 min-  40 min).

SECTION: Gel electrophoresis and immune-blotting: Running
32. Run samples through 10% gel (125 V, until front has reached bottom of gel, usually ~ 70 min).

SECTION: Gel electrophoresis and immune-blotting: Transfer
33. Remove gels from electrophoresis cell and construct transfer cassettes with PVDF membranes according to manufacturer’s instructions.
SECTION: Gel electrophoresis and immune-blotting: Transfer
34. Place the cassettes in the transfer cell and fill cell with Transfer buffer and icepack.
SECTION: Gel electrophoresis and immune-blotting: Transfer
35. Place the cell in a basin.
SECTION: Gel electrophoresis and immune-blotting: Transfer
36. Fill basin with ice around cell.
SECTION: Gel electrophoresis and immune-blotting: Transfer
37. Run transfer for 60 min- 70 min, 100 V.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
38. Dry membrane between filter paper in the dark for at least 45 min.
 

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
39. Rehydrate membrane in MetOH.
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
40. Wash in ddWater.
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
41. Wash in 1X TBS 2 min.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
42. Stain total protein, 5 min, Room temperature, with REVERT 700 Total Protein Stain.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
43. Wash membrane 2x  30 s with REVERT wash buffer.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
44.  Image total protein on LICOR.
 

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
45. Rinse in ddWater.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
46. Block membranes in EveryBlot buffer, 5 min, Room temperature with rocking.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
47. Incubate in vacuum packs with primary antibodies in EveryBlot  5 min at 4 °C.
 

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
48. Wash with TBST.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
49. Incubate with secondary antibody 1:20,000 in TrueBlack antibody diluent with 0.2% TWEEN and 1:1000 10% SDS for up to 60 min.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
50. Wash with TBST.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
51. Wash 1X with TBS to clear TWEEN.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
52. Wash 1X with water.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
53. Image
SECTION: Gel electrophoresis and immune-blotting: Quantification
54. For quantification of knockdown (Option 1).
SECTION: Gel electrophoresis and immune-blotting: Quantification
54.1. Use ImageStudio software to draw rectangles around total protein in each lane of Total Protein image and subtract background.
SECTION: Gel electrophoresis and immune-blotting: Quantification
54.2. Then add rectangles to outline p62 bands and subtract background.
SECTION: Gel electrophoresis and immune-blotting: Quantification
54.3. Transfer intensity measurements to Excel.
SECTION: Gel electrophoresis and immune-blotting: Quantification
54.4. Calculate p62 expression relative to total protein for each experiment.
SECTION: Gel electrophoresis and immune-blotting: Quantification
55. For quantification of p62 enrichment from GFP-NEMO immunoprecipitation.
SECTION: Gel electrophoresis and immune-blotting: Quantification
55.1. Use ImageStudio to add rectangles around input/cytosolic p62 and eluted p62.
SECTION: Gel electrophoresis and immune-blotting: Quantification
55.2. Transfer intensity measurements with background subtracted to Excel.
SECTION: Gel electrophoresis and immune-blotting: Quantification
55.3. Calculate p62 eluted relative to input amounts.
SECTION: Gel electrophoresis and immune-blotting: Quantification
55.4. Calculate elution/input quantity relative one condition.
 

SECTION: Gel electrophoresis and immune-blotting
10. 
 

SECTION: Gel electrophoresis and immune-blotting: Set-up
11.  Set up electrophoresis cell with 10% gels by manufacturer’s instructions.
 
SECTION: Gel electrophoresis and immune-blotting: Set-up
12.  Fill cell with RB and flush wells with a plastic transfer pipet.
SECTION: Gel electrophoresis and immune-blotting: Set-up
13.  Invert samples by hand to mix, and ensure all samples are at the bottom of tubes by briefly centrifuging.
SECTION: Gel electrophoresis and immune-blotting: Set-up
14. Load wells with equal amounts of protein (Option 1) or equal volumes (Options 2 and 3) and molecular weight standard (4 µL- 5 µL).
 



SECTION: Gel electrophoresis and immune-blotting: Set-up
15. For empty lanes, load approx. equal volume of 1X DB.
SECTION: Gel electrophoresis and immune-blotting: Running
16. Run samples through stacking gel (85 V, 20 min- 40 min).
 
SECTION: Gel electrophoresis and immune-blotting: Running
17.  Run samples through 10% gel (125 V, until front has reached bottom of gel, usually ~70 min).

SECTION: Gel electrophoresis and immune-blotting: Transfer
18. Remove gels from electrophoresis cell and construct transfer cassettes with PVDF membranes according to manufacturer’s instructions.
SECTION: Gel electrophoresis and immune-blotting: Transfer
19.  Place the cassettes in the transfer cell and fill cell with Transfer buffer and icepack.
SECTION: Gel electrophoresis and immune-blotting: Transfer
20. Place the cell in a basin.
SECTION: Gel electrophoresis and immune-blotting: Transfer
21. Fill basin with ice around cell.
SECTION: Gel electrophoresis and immune-blotting: Transfer
22. Run transfer for 60 min- 70 min, 100 V.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
23.  
Dry membrane between filter paper in the dark for at least 45 min.
 

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
24. Rehydrate membrane in MetOH.
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
25. Wash in ddWater.
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
26. Wash in 1X TBS 2 min.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
27. Stain total protein, 5 min, Room temperature, with REVERT 700 Total Protein Stain.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
28.  Wash membrane with REVERT wash buffer.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
29. Image total protein on LICOR.
 

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
30. Wash off total stain with REVERT Reversal (up to 10 min, Room temperature)

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
31. Rinse in ddWater.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
32. Block membranes in EveryBlot buffer, 5 min, Room temperature with rocking.
 
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
33. Incubate in vacuum packs with primary antibodies in EveryBlot  5 min at 4 °C.
 

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
34. Wash with TBST.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
35. Incubate with secondary antibody 1:20,000 in TrueBlack antibody diluent with 0.2% TWEEN and 1:1000 10% SDS for up to 60 min.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
36. Wash with TBST.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
37. Wash 1X with TBS to clear TWEEN.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
38. Wash 1X with water.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
39. Image
SECTION: Gel electrophoresis and immune-blotting: Quantification
40.  
For quantification of knockdown (Option 1).

SECTION: Gel electrophoresis and immune-blotting: Quantification
40.1. Use ImageStudio software to draw rectangles around total protein in each lane of Total Protein image and subtract
background.
SECTION: Gel electrophoresis and immune-blotting: Quantification
40.2. Then add rectangles to outline p62 bands and subtract background.
SECTION: Gel electrophoresis and immune-blotting: Quantification
40.3. Transfer intensity measurements to Excel.
SECTION: Gel electrophoresis and immune-blotting: Quantification
40.4. Calculate p62 expression relative to total protein for each experiment.
SECTION: Gel electrophoresis and immune-blotting: Quantification
41. For quantification of p62 enrichment from GFP-NEMO immunoprecipitation.
SECTION: Gel electrophoresis and immune-blotting: Quantification
41.1.  Use ImageStudio to add rectangles around input/cytosolic p62 and eluted p62.
SECTION: Gel electrophoresis and immune-blotting: Quantification
41.2. Transfer intensity measurements with background subtracted to Excel.
SECTION: Gel electrophoresis and immune-blotting: Quantification
41.3. Calculate p62 eluted relative to input amounts.
SECTION: Gel electrophoresis and immune-blotting: Quantification
41.4. Calculate elution/input quantity relative one condition.
 

SECTION: Gel electrophoresis and immune-blotting
33. 
 

SECTION: Gel electrophoresis and immune-blotting: Set-up
34. Set up electrophoresis cell with 10% gels by manufacturer’s instructions.
 
SECTION: Gel electrophoresis and immune-blotting: Set-up
35. Fill cell with RB and flush wells with a plastic transfer pipet.
SECTION: Gel electrophoresis and immune-blotting: Set-up
36. Invert samples by hand to mix, and ensure all samples are at the bottom of tubes by briefly centrifuging.
SECTION: Gel electrophoresis and immune-blotting: Set-up
37.  Load wells with equal amounts of protein (Option 1) or equal volumes (Options 2 and 3) and molecular weight standard (4 µL- 5 µL).
 



SECTION: Gel electrophoresis and immune-blotting: Set-up
38. For empty lanes, load approx. equal volume of 1X DB.
SECTION: Gel electrophoresis and immune-blotting: Running
39.  
Run samples through stacking gel (85 V, 20- 40 min).
 
SECTION: Gel electrophoresis and immune-blotting: Running
40. Run samples through 10% gel (125 V, until front has reached bottom of gel, usually ~ 70 min).

SECTION: Gel electrophoresis and immune-blotting: Transfer
41. Remove gels from electrophoresis cell and construct transfer cassettes with PVDF membranes according to manufacturer’s instructions.
SECTION: Gel electrophoresis and immune-blotting: Transfer
42. Place the cassettes in the transfer cell and fill cell with Transfer buffer and icepack.
SECTION: Gel electrophoresis and immune-blotting: Transfer
43. Place the cell in a basin.
SECTION: Gel electrophoresis and immune-blotting: Transfer
44. Fill basin with ice around cell.
SECTION: Gel electrophoresis and immune-blotting: Transfer
45. Run transfer for 60 min-  70 min, 100 V.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
46.  
Dry membrane between filter paper in the dark for at least  45 min.
 

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
47. Rehydrate membrane in MetOH.
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
48. Wash in ddWater.
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
49. Wash in 1X TBS 2 min.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
50. Stain total protein, 5 min, Room temperature, with REVERT 700 Total Protein Stain.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
51. Wash membrane with REVERT wash buffer.

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
52.  Image total protein on LICOR.
 

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
53.  Wash off total stain with REVERT Reversal (up to 10 min, Room temperature).

SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
54. Rinse in ddWater.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
55. 
Block membranes in EveryBlot buffer, 5 min,  Room temperature with rocking.
 
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
56. Incubate in vacuum packs with primary antibodies in EveryBlot   5 min at 4 °C.
 


SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
57. Wash with TBST.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
58. Incubate with secondary antibody 1:20,000 in TrueBlack antibody diluent with 0.2% TWEEN and 1:1000 10% SDS for up to 60 min.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
59.  Wash with TBST 4x 5 min.

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
60. Wash 1X with TBS to clear TWEEN.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
61. Wash 1X with water.
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
62.  Image
SECTION: Gel electrophoresis and immune-blotting: Quantification
63.  
For quantification of knockdown (Option 1).
 
SECTION: Gel electrophoresis and immune-blotting: Quantification
63.1. Use ImageStudio software to draw rectangles around total protein in each lane of Total Protein image and subtract
background.
SECTION: Gel electrophoresis and immune-blotting: Quantification
63.2. Then add rectangles to outline p62 bands and subtract background.
SECTION: Gel electrophoresis and immune-blotting: Quantification
63.3. Transfer intensity measurements to Excel.
SECTION: Gel electrophoresis and immune-blotting: Quantification
63.4. Calculate p62 expression relative to total protein for each experiment.
SECTION: Gel electrophoresis and immune-blotting: Quantification
64. For quantification of p62 enrichment from GFP-NEMO immunoprecipitation.
SECTION: Gel electrophoresis and immune-blotting: Quantification
64.1. Use ImageStudio to add rectangles around input/cytosolic p62 and eluted p62.
 
SECTION: Gel electrophoresis and immune-blotting: Quantification
64.2. Transfer intensity measurements with background subtracted to Excel.
SECTION: Gel electrophoresis and immune-blotting: Quantification
64.3. Calculate p62 eluted relative to input amounts.
SECTION: Gel electrophoresis and immune-blotting: Quantification
64.4. Calculate elution/input quantity relative one condition.

 

SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
34.1. Wash with TBST 4x 5 min (1/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
34.4. Wash with TBST 4x 5 min (4/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
34.3. Wash with TBST 4x 5 min (3/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
34.2. Wash with TBST 4x 5 min (2/4).
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
51.1. Wash membrane 2x 30 s with REVERT wash buffer (1/2).
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
51.2. Wash membrane 2x 30 s with REVERT wash buffer (2/2).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
57.1. Wash with TBST 4x 5 min (1/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
57.4. Wash with TBST 4x 5 min (4/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
57.3. Wash with TBST 4x 5 min (3/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
57.2. Wash with TBST 4x 5 min (2/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
59.1. Wash with TBST 4x 5 min (1/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
59.4. Wash with TBST 4x 5 min (4/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
59.3. Wash with TBST 4x 5 min (3/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
59.2. Wash with TBST 4x 5 min (2/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
48.1. Wash with TBST 4x  5 min (1/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
48.4. Wash with TBST 4x  5 min (4/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
48.3. Wash with TBST 4x  5 min (3/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
48.2. Wash with TBST 4x  5 min (2/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
50.1. Wash with TBST 4x  5 min (1/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
50.4. Wash with TBST 4x  5 min (4/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
50.3. Wash with TBST 4x  5 min (3/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
50.2. Wash with TBST 4x  5 min (2/4).
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
28.1.  Wash membrane 2x 30 s with REVERT wash buffer (1/2).
SECTION: Gel electrophoresis and immune-blotting: Membrane processing and total protein stain
28.2. Wash membrane 2x 30 s with REVERT wash buffer (2/2).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
36.1. Wash with TBST 4x 5 min (1/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
36.4. Wash with TBST 4x 5 min (4/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
36.3. Wash with TBST 4x 5 min (3/4).
SECTION: Gel electrophoresis and immune-blotting: Immuno-labeling
36.2. Wash with TBST 4x 5 min (2/4).