Anti-DYKDDDDK [M2] - Chimeric in Immunocytochemistry (HeLa)
Addgene #198002
- Data Submitted By
- Meghan Rego
- Lab Name
- Addgene
- Submission Date
- January 08, 2025
- Publication Date
- January 16, 2025 (modified January 17, 2025)
- Abstract
- Experimental results for Anti-DYKDDDDK [M2] - Chimeric (Addgene #198002) in an immunocytochemistry assay with HeLa cells transfected with rat Nrxn1-Flag.
![HeLa cells transfected with rat Neurexin-1-beta-Flag were fixed and labeled with Anti-Neurexin-1-Beta [N170A/1.1R] (green; Addgene #180100) and Anti-DYKDDDDK [M2] - Chimeric (red; Addgene #198002). Nuclei were counterstained with DAPI.](https://datahub-media.addgene.org/report-images/teb2eh/Nxn1.png)
Image attribution: Meghan Rego, Addgene
This report is made available under the Creative Commons Attribution 4.0 International License.
- Antibodies Used
-
Anti-DYKDDDDK [M2] - Chimeric
Addgene #198002 -
Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594
Thermo Fisher #A-11012
Materials & Methods
Sample
- Species
- Rat
- Cell / Tissue Type
- HeLa
- Additional Information
- Transiently transfected with rat Nrxn1 (Addgene #197339)
Buffer
- Name
- PBS
- pH
- 7.4
Fixative
- Fixative
- Formaldehyde/Paraformaldehyde
- Time
- 15 min
- Temperature
- 4 ˚C
Permeabilization
- Agent
- PBS containing 0.2% Triton X-100
- Time
- 10 min
- Temperature
- Room Temperature
Blocking
- Agent
- BSA in PBS containing 0.3% Triton X-100
- Concentration
- 5% w/v
- Time
- 60 min
- Temperature
- Room Temperature
Primary Antibody
- Name
- Anti-DYKDDDDK [M2] - Chimeric
- Source
- Addgene
- Catalog Number
- 198002
- Species
- Rabbit
- Gene / Target
- DYKDDDDK (Flag tag)
- Concentration
- 0.1 µg/mL
- Time
- 60 min
- Temperature
- Room Temperature
Secondary Antibody
- Name
- Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594
- Source
- Thermo Fisher
- Catalog Number
- A-11012
- Concentration
- 2 µg/mL
- Time
- 45 min
- Temperature
- Room Temperature
Additional Information
- Additional Information
HeLa cells were transfected with 0.1 µg of plasmid Nrxn-1 beta Flag/pCMV (Addgene #197339).
Cells were fixed and labeled 96 h later with antibodies against Neurexin-1-beta (Nrxn1; Addgene #180100) and Flag (Addgene #198002). Nuclei were counterstained with DAPI.
Permeabilization and blocking were performed with gentle agitation. Primary and secondary antibodies were diluted in PBS containing 0.3% Triton X-100 and 1% w/v BSA.
The secondary antibody used for detection of Anti-Neurexin-1-Beta was the goat anti-mouse IgG2a cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher, A-21131).
- Protocol
- Addgene immunocytochemistry protocol
- Controls
- Untransfected cells serve as the negative control and colocalization of the Nrxn1 and Flag signals in transfected cells serves as a positive control.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- Observed colocalization of the Nrxn1 (green) and Flag (red) signals in transfected cells, and no signal in the untransfected cells.