ICC/IF for Anti-Glypican 6 (GPC6) [IPI-GPC6.6] in specificity assay
Addgene #241001
- Data Submitted By
- Alexander Morano , Travis Riedel and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- October 03, 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) specificity assay with ExpiCHO cells expressing human and mouse GPCs.
![(Top) Immunofluorescence (IF) of ExpiCHO cells transfected with human and mouse GPC1, GPC2, GPC3, GPC4, GPC5, and GPC6. Human GPC targets were co-transfected with GFP (human transfection control) and mouse GPC targets were co-transfected with BFP (mouse transfection control). Cells were labeled with Anti-Glypican 6 (GPC6) [IPI-GPC6.6] (Addgene #241001) and imaged for GFP (green), BFP (blue), and GPC6 (magenta) at 10X magnification. (Bottom) Each graph depicts the combined quantification of multiple images of the same transfected cells taken at 10X magnification. IPI-GPC6.6 labeling of human and mouse variants of each GPC family member is compared on the top and bottom rows. For all panels, IPI-GPC6.6 was used at 1 μg/mL (1:1,000 dilution).](https://datahub-media.addgene.org/report-images/bx02yk/GPC6.6_IF_Specificity.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 separately transfected with human and mouse GPC1, GPC2, GPC3, GPC4, GPC5, and GPC6. Human GPCs were cotransfected with GFP, and mouse GPCs were cotransfected with BFP.
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 10X 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 (not shown): 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 (not shown): House-made rabbit anti-mOPC2 antibody used to confirm transfected construct does not interact /produce signal with every antibody.
Negative control 2 (not shown): Testing antibody against transfected control construct (CD276) containing IGG domains to ensure nonspecific binding is not occurring.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- The antibody is specific to human and mouse GPC6 and does not react with other GPC family members.