TITLE: Cell lysis and gel electrophoresis for protein analysis of HeLa cells (Provisional unformatted)
AUTHORS: OLIVIA   HARDING, 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 co-localization with p62 is also dependent on multiple factors.

[STEPS]
1. -       This protocol was developed to analyze protein expression and enrichment in cell culture, including HeLa-M cells and HeLa p62-/- cells
2. -       Option 2 was modified from a protocol used in Turco et al, Molec. Cell, 2019
3. -       Option 3 was modificed from a protocol used in Wurzer et al, eLife, 2015  Materials
4. -      
5. 5 mL capped tubes (Millipore Sigma, EP022364120)
6. -       Cell scrapers
7. -       Liquid nitrogen
8. -      
9. 5 mL Lo-Bind capped tubes (Eppendorf, 022431081)
10. -       10% acrylamide gels with desired number of wells (make or purchase)  Reagents  For all Options
11. -       1X Phosphate buffered saline (PBS)
12. -       2% bovine serum albumin (BSA) (Fisher Scientific, BP1605-100) for Option 2
13. -       10% sodium dodecyl sulfate solution (SDS) (Invitrogen, 15553-035)
14. -       Protease and phosphatase inhibitors
15. o   Leupeptin (10 mg/mL = 1000X)
16. o   DTT (1M = 1000X)
17. o   Pepstatin A (1 mg/mL = 1000X)
18. o   TAME (10 mg/mL = 1000X)
19. o   PMSF (100mM = 100X)
20. -       4X Denaturing buffer (DB) [900 uL (4% SDS, 50% glycerol, 125 mM Tris HCl, pH 6.8, 0.2% w/v Orange G) + 100 uL beta-mercaptoethanol (BME)]
21. -       Methanol
22. -       4X Running buffer (RB)
23. -       1X Tris buffered saline (TBS)
24. -       1X TBS with 0.1% Tween (BIO-RAD, 1706531) (TBST)
25. -       Running Buffer (RB) (250 mL 4X RB + 750 mL water + 10 mL 10% SDS)
26. -       Transfer buffer (125 mL 4X RB + 775 mL water +
27. mL methanol + 500 uL 10% SDS + 560 uL BME)
28. -       PVDF membranes
29. -       REVERT 700 Total Protein Stain (LICOR, 926-11021)
30. -       REVERT Wash Buffer (6.7% w/v glacial acetic acid, 30% v/v methanol in water)
31. -       REVERT Reversal Buffer (0.1 M NaOH, 30% v/v methanol in water)
32. -       Desired primary antibodies
33. -       LICOR secondary antibodies such as donkey anti-mouse 800 (926-32212) and donkey anti-rabbit 680 (926-68073)
34. -       TrueBlack buffer (Biotium, 23013B-1L)
35. -       EveryBlot buffer (BIO-RAD, 12010020)     Specialized buffers and other reagents
36. -       RIPA buffer (Option 1)     Reagent   stock concentration   final concentration   volume of stock (for 10 mL)     Tris-HCl (pH 8.0)
37. M
38. mM
39. uL     EDTA
40. mM
41. mM
42. uL     EGTA
43. mM
44. mM
45. uL     Triton X-100   10%   1%
46. uL     DOC   5%
47. 50%
48. uL     SDS   10%
49. 10%
50. uL     NaCl
51. M
52. mM
53. uL     Water   -   -
54. mL
55. -       Lysis Buffer-A (Option 2)       Reagent   Final conc   For 10 mL
56. M HEPES/KOH
57. mM pH 7.5
58. uL
59. M Sorbitol
60. mM
61. 5 mL
62. mM EGTA
63. 5 mM
64. uL
65. M Mg-Acetate
66. mM
67. uL     ddH2O    
68. 92 mL
69. -       PBS with 0.1% TWEEN (PBST) for Option 2
70. -       Wash Buffer (Option 3)       Reagent   Final conc   For 20 mL
71. M Tris-Cl
72. mM, pH 7.4
73. uL     100% Glycerol   10%
74. mL
75. M NaCl
76. mM
77. uL     ddH2O    
78. 06 mL
79. -       Lysis Buffer-B (Option 3)       Reagent   Final conc   For 5 mL     Master buffer
80. mM, pH 8.0
81. 975 mL     100% NP-40 (IGEPAL)
82. 5%
83. uL        
84. -       Pierce BCA Assay (ThermoFisher, 23225) for Option 1
85. -       GFP-Trap Magnetic Particles (Chromotek M-270) for immuno-precipitations  Equipment
86. -       vacuum apparatus
87. -       end-on-end rotating apparatus
88. -       refrigerated centrifuge
89. -       magnetic rack (for GFP-Trap particle precipitation)
90. -       rockers at room temperature (R.T.) and at 4C
91. -       Plate reader (such as BioTex Synergy Mx)
92. -       Odyssey CLx membrane scanner (LI-COR, 9140)
93. -       ImageStudio software (LI-COR)
94. -       Heat source to 95 C
95. -       Gel electrophoresis apparatus (BIO-RAD)
96. -       Membrane transfer apparatus (BIO-RAD)
97. -       Excel  Before start
98. -       The start point for this protocol is after cells grown on 3.5 cm, or 10 cm dishes have been transfected with relevant constructs for 18-24 hours and treated with appropriate small molecules or vehicles
99. -       For 3.5 cm dishes, follow transfection procedures enumerated in imaging protocols.
100. -       Chill all reagents on ice
101. -       Add protease and phosphatase inhibitors to 1X to each lysis buffer immediately before use  Step-by-step  Wash cells
102. -       Aspirate media from dishes
103. -       Wash samples quickly x2 with ice cold PBS
104. o   Note: Can stop after washes by scraping cells with the second wash of PBS into 1.5 mL tube, spin down at 2.4G, 3min, 4 degrees. Aspirate PBS and snap-freeze tubes in liquid nitrogen. If frozen, add respective lysis buffer and inhibitors and let thaw on ice 10 min before proceeding  Option 1: Standard lysis with RIPA  Note: We used this protocol to assess depletion of p62 in HeLa-M cells after siRNA treatment and imaging NEMO recruitment. Samples were collected from 35 mm imaging dishes.
105. -       Add
106. uL RIPA + inhibitors to dish and scrape cells into 1.5 uL tube, OR add buffer to thawed sample and resuspend by pipetting
107. -       Rotate resuspended sample on end-over-end machine at 4 degrees for 20 min
108. -       Spin at top speed (17 G), 4 degrees, 20 min
109. -       Remove supernatant as Lysis and keep on ice or store at -80 C
110. -       Measure protein concentration with Pierce BCA assay by adding 25 uL sample or BSA standard to each well in duplicate and 200 uL Reagent A+B. Incubate 37 C for 30 min then measure absorbance on a plate reader
111. o   Note: it is likely necessary to dilute samples 1:4 or more to measure within the range of the assay.
112. -       Add 1/3 volume of 4X DB to remaining Lysis or a measured fraction of sample and heat 95 C for 5 min
113. -       Proceed to gel electrophoresis  Option 2: Co-IP EGFP-NEMO for p62 association assay in AntA/OligA or vehicle conditions
114. -       The samples for this assay are: A) EGFP-vector + veh, B) EGFP-vector + AntA/OligA, C) EGFP-NEMO + veh, and D) EGFP-NEMO + AntA/OligA. Start with 3x 10 cm dish per condition for one biological replicate
115. -       18-24 hr before collection, transfect 3 ug Parkin and 1.5 ug EGFP-NEMO (or vector), to 70-80% confluent cells on each dish
116. -       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  Lysis with heavy fraction isolation
117. -       Scrape and combine like samples into 800 uL PBS, transfer to 1.5 mL tube
118. -       Spin
119. min, 2 G, at 4 C
120. -       Aspirate PBS and snap freeze in liquid nitrogen
121. -       Add
122. uL LB-A with inhibitors to each frozen sample
123. -       Pipet to resuspend
124. -       Incubate on ice 10 min
125. -       centrifuge at 1000g, 5 min, 4 degrees
126. -       Transfer sup. (Cytosol) to a new tube
127. -       Centrifuge again at top speed, 10 min, 4 degrees
128. -       Save pellet as Heavy Fraction if desired and resuspend in 50 uL 1X DB  Prep IP beads
129. -        Mix GFP-Trap beads by swirling and gently pipetting to break up accumulations
130. -        aliquot 25 uL bead slurry into LoBind tubes (one for each condition)
131. -        Remove supernatant from beads by inserting the tube in a magnetic rack and carefully aspirating clear solution
132. -        Resuspend beads in 500 uL cold PBST
133. -        Insert to magnetic rack and remove PBST wash
134. -        Block beads in 2% BSA (in water) for 1 hr at 4 degrees with end-over-end rotation
135. -        wash 2X with 500 uL r.t. PBST
136. -        wash 1X with 500 uL LB-A
137. -        Allow magnets to collect beads for up to 5 min  Bead capture
138. -       Add
139. uL Cytosol to bead tube, rotate end-over-end gently for 1 hr, 4 degrees (save the rest of Cytosol for Input)
140. -       Remove supernatant by magnet (up to 5 min) and save as Flow Through
141. -       Wash 3X with 500 uL R.T. PBS (no TWEEN) (can save these washes)
142. -       for the last wash, transfer beads and wash to a new tube
143. o   Note: optional if using Lo-bind tubes
144. -       Remove sup from last wash and resuspend in ~90 uL 1X DB with rough mixing (vortex, pipetting), then heat 95 C for 5 min
145. -        Magnetize 5 min and save sup as Eluate
146. -        Add 1/3 volume of 4X DB to Input and Flow Through and heat 95 C for 5 min
147. -        Proceed to gel electrophoresis  Option 3: Co-IP GFP-Ubiquitin for p62 and NEMO association assay
148. -       The samples for this assay are in p62-/- Hela cells: A) EGFP-vector + mCherry-vector, B) EGFP-vector + mCherry-p62, C) EGFP-Ubiquitin + 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
149. -       18-24 hr before collection, transfect 3 ug Parkin and EGFP-Ubiquitin (or vector), 1.5 ug NEMO, and 2-2.5 ug mCherry-p62 (or vector) to 70-80% confluent cells in each dish
150. -       Note: 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  Lysis
151. ·       Combine samples by scraping cells into 600 uL LB-B with inhibitors and transfer to 1.5 mL tube
152. ·       Incubate on ice, 15 min
153. ·       Centrifuge at 16,100 g (16.1 G), 13 min, 4 degrees
154. ·       Transfer sup. to a new tube as Lysis     Prep IP beads
155. ·       Mix GFP-Trap beads by swirling or gently pipetting to break up accumulations and aliquot 10 uL into LoBind tubes (one for each condition)
156. ·       Remove supernatant from beads by inserting the tube in a magnetic rack and carefully aspirating clear solution
157. ·       Resuspend beads in 500 uL r.t. Wash Buffer
158. ·       Insert to magnetic rack and remove wash
159. ·       Repeat for a total of 2 washes
160. ·       wash 1X with 500 uL LB-B
161. ·       Allow magnets to collect beads for up to 5 min     Bead capture
162. ·       Add 500 uL Lysed sample to bead tube, rotate gently for 1 hr, R.T.
163. ·       save the rest of the Lysis as Input
164. ·       Remove sup by magnet (up to 5 min) and save as Flow Through
165. ·       Wash 3X with 500 uL R.T. Wash buffer (can save these washes)
166. ·       for the last wash, transfer beads and wash to a new tube (optional if you’re using Lo-bind tubes)
167. ·       Remove sup from last wash and resuspend in ~90 uL 1X DB with rough mixing (vortex, pipetting), then heat block 10 min
168. ·       Magnetize 5 min to collect beads and save sup as Eluate
169. ·       Add 1/3 volume of 4X DB to Input and Flow Through and heat 95 C for 5 min
170. ·       Proceed to gel electrophoresis  Gel electrophoresis and immune-blotting
171. -       This protocol was developed for use with the LI-COR system for protein detection  Set-up
172. -       Set up electrophoresis cell with 10% gels by manufacturer’s instructions
173. -       Fill cell with RB and flush wells with a plastic transfer pipet.
174. -       Invert samples by hand to mix, and ensure all samples are at the bottom of tubes by briefly centrifuging
175. -       Load wells with equal amounts of protein (Option 1) or equal volumes (Options 2 and
176. and molecular weight standard (4-5 uL).
177. o   Note: We load 15-25 uL eluate and 10 uL Input
178. -       For empty lanes, load approx. equal volume of 1X DB.  Running
179. -       Run samples through stacking gel (85 V, 20-40 min)
180. -       Run samples through 10% gel (125 V, until front has reached bottom of gel, usually ~70 min)     Transfer
181. -       Remove gels from electrophoresis cell and construct transfer cassettes with PVDF membranes according to manufacturer’s instructions
182. -       Place the cassettes in the transfer cell and fill cell with Transfer buffer and icepack
183. -       place the cell in a basin
184. -       Fill basin with ice around cell
185. -       Run transfer for 60-70 min, 100 V  Membrane processing and total protein stain
186. -       Dry membrane between filter paper in the dark for at least 45 min
187. o   Note: this is most important for small proteins
188. o   Note: can be a stopping point for several days
189. -       Rehydrate membrane in MetOH
190. -       Wash in ddWater
191. -       Wash in 1X TBS 2 min
192. -       Stain total protein, 5 min, R.T., with REVERT 700 Total Protein Stain
193. -       Wash membrane 2x 30 sec with REVERT wash buffer
194. -       Image total protein on LICOR
195. o   Note: can cut the membrane based on total stain if desired
196. -       Wash off total stain with REVERT Reversal (up to 10 min, R.T.)
197. -       Rinse in ddWater  Immuno-labeling
198. -       Block membranes in EveryBlot buffer, 5 min, R.T. with rocking
199. -       Incubate in vacuum packs with primary antibodies in EveryBlot overnight at 4 C. See materials and methods for concentrations of antibodies used.
200. -       Wash with TBST 4x 5 min
201. -       Incubate with secondary antibody 1:20,000 in TrueBlack antibody diluent with 0.2% TWEEN and 1:1000 10% SDS for up to 1 hr
202. -       Wash with TBST 4x 5 min
203. -       Wash 1X with TBS to clear TWEEN
204. -       Wash 1X with water
205. -       Image   Quantification
206. -       For quantification of knockdown (Option 1)
207. o   Use ImageStudio software to draw rectangles around total protein in each lane of Total Protein image and subtract background
208. o   Then add rectangles to outline p62 bands and subtract background
209. o   Transfer intensity measurements to Excel
210. o   Calculate p62 expression relative to total protein for each experiment
211. -       For quantification of p62 enrichment from GFP-NEMO immuno-precipitation
212. o   Use ImageStudio to add rectangles around input/cytosolic p62 and eluted p62
213. o   Transfer intensity measurements with background subtracted to Excel
214. o   Calculate p62 eluted relative to input amounts
215. o   Calculate elution/input quantity relative one condition
216. §  Note: we found it necessary to perform this secondary normalization due to variability across replicates. In our case, we normalized results from each condition to the EGFP-NEMO + AntA/OligA results