Jun 15, 2023

Public workspaceWestern Blotting

DOI
https://dx.doi.org/10.17504/protocols.io.36wgq3rzklk5/v1
  • Eric Cordeiro-Spinetti1
  • 1University of Miami
Eric ECS Cordeiro-Spinetti
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgq3rzklk5/v1
Protocol Citation: Eric Cordeiro-Spinetti 2023. Western Blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3rzklk5/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: June 15, 2023
Last Modified: June 15, 2023
Protocol Integer ID: 83418
Keywords: western blotting
Abstract
It's a classic
Troubleshooting
Before Starting
2h 40m 45s

Make RIPA buffer (to be used in 3 months after adding Nuclease)
AB
50mM Tris HCl, PH 7.4 5mL (1M stock)
150mM NaCl 5mL (3M stock)
1% Triton X-100 or NP-40 1 mL
0.1% SDS 1mL (10% supply center solution)
1mM EDTA 0.2 mL (0.5M stock)
10mM Naf 0.042g (@L5P1 chemical room)
0.5% Sodium deoxylcholate 0.5g (@L5P1 chemical room)
Add ddH2O to 100 mL
Add PMSF [final] = 1mM and any other protease inhibitors immediately before use
Add 1uL Pierce™ Universal Nuclease per 1mL


SDS Running Buffer
Cell lysis
Trypsinize cells
Wash pellet with PBS
Remove all supernatant with micropipette
Freeze pellet in liquid N2
Thaw quickly at Temperature37 °C

Resuspend cell pellet on buffer Amount100 µL

Vortex Duration00:00:15

15s
Incubate on Ice Duration00:20:00

20m
Vortex Duration00:00:15

Collect supernatant
Centrifuge 10000 RPM for Duration00:05:00

5m
Running, Transfer and Blot
2h 40m 45s
Collect supernatant without disturbing the pellet
Quantify protein
799 uL water + 1 uL lysate
+ 200 uL 5x Bradford reagent
Read at 595 nm
Load 50 µg of protein/well
Loading buffer Amount50 µL β-mercaptoethanol + Amount950 µL 2x Laemli Buffer

Water up to Amount15 µL

Boil atTemperature95 °C before loading  Duration00:00:15

15s
Add Amount10 µL of ladder (Precision Plus Protein - Dual Color Standards)

Run at 120-150 volts Duration01:00:00

1h
Activate PVDF membrane in methanol
Soak filter paper in Towbin Buffer
Equilibrate gel in Towbin buffer Duration00:10:00

10m
Semi-dry transfer
90' (<50KDa) or 120' (>50KDa)
Mount sandwich (paper-membrane-gel-paper)
45 mA/gel (constant current)
Cut out LEFT UPPER corner of the membrane
Block membrane with 5% BSA in PBS 0.1% Tween (PBST) for Duration00:30:00

30m
Add primary antibody in BSA-PBST and incubate overnight Duration18:00:00 at Temperature4 °C

18h
Wash 3x with PBST for Duration00:05:00

5m
Add HRP secondary antibody in BSA-PBST and incubate forDuration01:00:00 at room temperature on a shaker

1h
Wash 3x with PBST forDuration00:05:00

5m
Add 500 µL ECL (enhanced chemilumescent) per membrane strip
Protocol references
https://www.ptglab.com/news/blog/lysate-preparation-why-is-ripa-buffer-best-for-western-blot/

https://docs.abcam.com/pdf/protocols/sample-preparation-for-western-blot.pdf
RIPA buffer is useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X-100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations and pull-down assays.

https://www.usbio.net/protocols/immunoprecipitation


  • We recommend using 1.0 mL of RIPA Lysis Buffer to lyse 0.5 to 5 x 10E7 adherent mammalian cells.
  • 1mL of buffer per 75cm2 flask containing 5 × 10ˆ6 HeLa or A431 cells.


RIPA buffer 100mL
50mM Tris HCl, PH 7.4 5mL (1M stock)
150mM NaCl 5mL (3M stock)
1% Triton X-100 or NP-40 1 mL
0.1% SDS 1mL (10% supply center solution)
1mM EDTA 0.2 mL (0.5M stock)
10mM Naf 0.042g (@L5P1 chemical room)
0.5% Sodium deoxylcholate 0.5g (@L5P1 chemical room)
Add ddH2O to 1000ml
Add PMSF to a final concentration of 1mM and any other protease inhibitors immediately before use.
Add 1 uL Pierce™ Universal Nuclease per 1mL




4 x SDS sample buffer For 1000ml
12% SDS 120g
25% Glycerol 250ml
150 mM Tris.HCl (pH 7.0 1M stock) 150ml
0.03% Bromophenol Blue 300mg
20% β-mercaptoethanol 200ml
Add ddH2O to 50ml, aliquot and store at -20°C
20% β-mercaptoethanol, (or 500 mM DTT replaced) should be added freshly before use.


Nonidet P-40 (IGEPAL® CA-630) 0.5%


Pierce™ Universal Nuclease for Cell Lysis added to RIPA NP40 1% buffer
0.1uL nuclease per 1 mL buffer = 25U/mL [final]
https://assets.thermofisher.com/TFS-Assets/LSG/certificate/Certificates-of-Analysis/00492617_88700.pdf