Jun 04, 2025
Western blot - HA (Cell Signaling, #51872S) and anti-TBK1 to distinguish endogenous vs overexpressed TBK1
- 1NIMH
Protocol Citation: Jailyn Izu 2025. Western blot - HA (Cell Signaling, #51872S) and anti-TBK1 to distinguish endogenous vs overexpressed TBK1. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l627o5gqe/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: July 09, 2024
Last Modified: June 04, 2025
Protocol Integer ID: 103100
Funders Acknowledgements:
National Institute of Mental Health
Grant ID: ZIA MH002982
National Institute of Mental Health
Grant ID: R01MH122471
National Institute of Neurological Disorders and Stroke
Grant ID: U01NS120836
Foundation for the National Institutes of Health
Grant ID: Deeda Blair Research Initiative for Disorders of the Brain
Abstract
Western blot to distinguish endogenous vs overexpressed TBK1
Materials
- 4-12% Bis-Tris (Invitrogen NuPAGE-SDS) mini gels (1.0 mm x 10 well or 1.5 mm x 15 well) (Invitrogen, #NP0321BOX /#NP0323BOX)
- MOPS-SDS running buffer (Invitrogen, #NP0001)
- 4x LDS buffer (Invitrogen, #NP0007)
- 10x dithiothreitol/DTT (Thermo Fisher Scientific, #A39255)
- Li-Cor Chameleon Duo Pre-stained Protein Ladder (Li-Cor, P/N: 928-60000)
- Tris Glycine transfer buffer 10X (KD Medical, #RGF-3391)
- HPLC Methanol (Fisher Scientific, #A452SK)
- Li-Cor Intercept (TBS) blocking buffer (Li-Cor, P/N: 927-60001)
- Tris-buffered saline 10X (Corning, #46-012-CM)
- Tween20 (Sigma, #P1379)
- Sodium azide (Sigma, #S-8032)
- HA primary antibody (Cell Signaling, #3724S)
- TBK1 primary antibody (Cell Signaling, #51872S)
- Beta actin antibody (Cell Signaling, #4970S)
- IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (Li-Cor, P/N: 926-32210)
- IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (Li-Cor, P/N: 926-68071)
- Immun-Blot LF PVDF Membrane (BioRad, #1620264)
- Filter papers (Whatman, #1001125)
- XCell II Blot Module (Invitrogen, #EI9051)
- 1.5 mL eppendorf tubes
- Transfer sponges
- Aluminum foil
- Li-COR Odyssey M
Western blot
Western blot
Follow the general western blot protocol:
Protocol-specific parameters:
- For Step 3, load 3 uL lysate
- Skip Steps 11-12 (normalizing to beta-actin instead of total protein)
- Note: before Step 14, you will cut the membrane with the transferred proteins at the 50 kDa mark and probe the top (Gel 1/1) and bottoms (Gel 1/2) with separate primary and secondary antibodies.
- For Gel 1/1 for the HA primary antibody (Cell Signaling, #3724S) in Step 14, use a 1:1000 dilution (v/v) in blocking buffer: 6 mL Licor blocking buffer solution + 6 μl Tween20 (0.1%) + 6 µL HA primary antibody
- For Gel 1/1 for the TBK1 primary antibody (Cell Signaling, #51872S) in Step 14, use a 1:1000 dilution (v/v) in blocking buffer: 6 mL Licor blocking buffer solution + 6 μl Tween20 (0.1%) + 6 µL TBK1 primary antibody
- For Gel 1/2 for the beta actin antibody (Cell Signaling, #4970S) in Step 14, use a 1:6000 dilution (v/v) in blocking buffer: 6 mL Licor blocking buffer solution + 6 μl Tween20 (0.1%) + 1 µL beta actin primary antibody
- For Step 17, see materials for secondary antibodies