ICC/IF for Anti-Glypican 6 (GPC6) [IPI-GPC6.6] in binding assay
Addgene #241001
- Data Submitted By
- Alexander Morano , Travis Riedel and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- September 26, 2025
- Publication Date
- October 03, 2025 (modified October 22, 2025)
- Abstract
- Experimental results for Anti-Glypican 6 (GPC6) [IPI-GPC6.6] (Addgene #241001) in an immunofluorescence (IF) assay with ExpiCHO cells expressing human or mouse GPC6.
![A) Immunofluorescence (IF) of ExpiCHO cells transfected with V5-tagged human and mouse GPC6. Human GPC6 was co-transfected with GFP (human transfection control) and mouse GPC6 was co-transfected with BFP (mouse transfection control). Cells were transfected separately and mixed for labeling and imaging. Cells were labeled with Anti-Glypican 6 (GPC6) [IPI-GPC6.6] (Addgene #241001) and imaged for GFP (green), BFP (blue), and GPC6 (magenta) at 40X magnification. B) Combined quantification of multiple images of the same transfected cells taken at 10X magnification. IPI-GPC6.6 labeling of human (left) and mouse (right) GPC6 are shown in the top row, and compared to a positive (left) and negative (right) control in the bottom row. For both panels, IPI-GPC6.6 was used at 1 μg/mL (1:1,000 dilution).](https://datahub-media.addgene.org/report-images/5pok37/GPC6.6_IF_Binding.png)
Image attribution: Institute for Protein Innovation (IPI)
This report is made available under the Creative Commons Attribution 4.0 International License.
- Antibodies Used
-
Anti-Glypican 6 (GPC6) [IPI-GPC6.6]
Addgene #241001 -
Alexa Fluor 647 F(ab')2 fragment goat anti-rabbit IgG, Fc fragment-specific
Jackson ImmunoResearch #111-606-046
Materials & Methods
Sample
- Target Species
- Human
- Cell / Tissue Type
- ExpiCHO
- Additional Information
- ExpiCHO cells were transiently transfected with GFP (transfection control) and human GPC6
Buffer
- Name
- PBS
Fixative
- Fixative
- Formaldehyde/Paraformaldehyde
- Time
- 15 min
- Temperature
- Room Temperature
Permeabilization
- Agent
- Triton X-100 in PBS
- Concentration
- 0.20%
- Time
- 10 min
- Temperature
- Room Temperature
Blocking
- Agent
- Normal goat serum + BSA
- Concentration
- 5% serum, 5% BSA
- Time
- 30 min
- Temperature
- Room Temperature
Primary Antibody
- Name
- Anti-Glypican 6 (GPC6) [IPI-GPC6.6]
- Source
- Addgene
- Catalog Number
- 241001
- Host Species
- Rabbit
- Target Antigen
- GPC6
- Concentration
- 1 µg/mL
- Time
- 60 min
- Temperature
- Room Temperature
Secondary Antibody
- Name
- Alexa Fluor 647 F(ab')2 fragment goat anti-rabbit IgG, Fc fragment-specific
- Source
- Jackson ImmunoResearch
- Catalog Number
- 111-606-046
- Concentration
- 2 µg/mL
- Time
- 60 min
- Temperature
- Room Temperature
Additional Information
- Additional Information
Confocal water-immersion images taken at 40X magnification on the ImageXpress confocal HT.ai microscope.
Widefield 10X magnification for quantitative data. GFP- or BFP-positive cells were identified using a custom module in the IN Carta image analysis software, then the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing Anti-Glypican 6 (GPC6) [IPI-GPC6.6] labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 450 (background fluorescence) to 20000.
Controls
Positive control: Anti-V5 [IPI-SV5-Pk1] (Addgene #218107) used to confirm IF labeling efficacy (since construct expressing target also has a V5 tag).
Negative control 1: House-made rabbit anti-mOPC2 antibody used to confirm transfected construct does not interact/produce signal with every antibody.
Negative control 2: Testing antibody against transfected control construct (CD276) containing IGG domains to ensure nonspecific binding is not occuring.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- The antibody shows binding to human and mouse GPC6.