Jun 04, 2025
Western blot - EGFP (Invitrogen, #A10262) protein expression
- 1NIMH
Protocol Citation: Jailyn Izu 2025. Western blot - EGFP (Invitrogen, #A10262) protein expression. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19o2olr2/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: May 15, 2024
Last Modified: June 04, 2025
Protocol Integer ID: 99869
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 for detected overexpressed EGFP in COS1 cell lysate
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)
- Antibodies: EGFP (Invitrogen, #A10262)
- Sodium azide (Sigma, #S-8032)
- 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), IRDye 800CW Donkey anti-Chicken Secondary Antibody (Li-Cor, P/N: 926-32218)
- 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 total protein loading amounts in Step 3, load 5 µg of total protein for each sample (calculate from Bradford Assay)
- Skip Steps 11 & 12 (normalizing to ERK1 instead of total protein)
- For the EGFP primary antibody (Invitrogen, #A10262) 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 EGFP primary antibody