ICC/IF for Rabbit IgG Isotype Control [IPI-MOPC-21] in binding assay
Addgene #237822
- Data Submitted By
- Alexander Morano , Travis Riedel and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- March 25, 2026
- Publication Date
- March 27, 2026 (modified April 13, 2026)
- Abstract
- Experimental results for Rabbit IgG Isotype Control [IPI-MOPC-21] (Addgene #237822) in an immunofluorescence (IF) binding assay with ExpiCHO cells expressing human or mouse V5-tagged Semaphorin 4B.
![A) Immunofluorescence (IF) of ExpiCHO cells transfected with human (Green/GFP) or mouse (Blue/BFP) SEM4B-V5 labeled in magenta with IPI-MOPC21 (top row) and Anti-V5 [IPI-SV5-Pk1] (bottom row, Addgene #218107). Confocal images taken at 40X magnification on the ImageXpress confocal HT.ai microscope. Scale bar: 20 μM. B) Combined quantification of multiple images of the same transfected cells taken at 10X magnification. GFP- or BFP-positive cells were identified via the neural network CellPose, then the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing IPI-MOPC21 or IPI-SV5-Pk1 labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 500 (background fluorescence) to 22000. IPI-MOPC21 labeling of human and mouse SEM4B is shown in the top row, and compared to the positive control (IPI-V5 labeling) in the bottom row. For both panels, IPI-MOPC21 was used at 1:1000 (1 μg/mL)](https://datahub-media.addgene.org/report-images/6tylmq/MOPC21-IF-Binding_v2.png)
Image attribution: Institute for Protein Innovation (IPI)
This report is made available under the Creative Commons Attribution 4.0 International License.
- Antibodies Used
-
Rabbit IgG Isotype Control [IPI-MOPC-21]
Addgene #237822 -
Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
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 V5-tagged Semaphorin 4B.
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
- Rabbit IgG Isotype Control [IPI-MOPC-21]
- Source
- Addgene
- Catalog Number
- 237822
- Host Species
- Rabbit
- Concentration
- 1 µg/mL (1:1,000 dilution)
- Time
- 60 min
- Temperature
- Room Temperature
Secondary Antibody
- Name
- Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
- Source
- Jackson ImmunoResearch
- Catalog Number
- 111-606-046
- Concentration
- 2 µg/mL
- Time
- 60 min
- Temperature
- Room Temperature
Additional Information
- Additional Information
Confocal images taken at 40X magnification on an ImageXpress confocal HT.ai microscope.
For quantitative data, GFP- or BFP-positive cells were identified via the neural network CellPose, then the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing Rabbit IgG Isotype Control [IPI-MOPC-21] labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 100 (background fluorescence) to 22000 (saturation).
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: Rabbit IgG used to confirm lack of nonspecific binding to the sample.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- The antibody does not show nonspecific binding in the sample or to either of the selected targets.