Protein Block (ab64226)
Overview
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Product name
Protein Block -
Tested applications
Suitable for: IHC-P, ELISA, WBmore details -
General notes
Protein blocking buffer ab64226 for serum-free blocking of non-specific antibody binding in IHC, ELISA and western blot. Ready to use. No mixing or dilution required.
For use in ELISA, add Protein Block to well and incubate for 2-10 minutes before addition of sample. Wash and continue procedure. For use in western blotting, incubate for one hour at room temperature or overnight at 4°C.
IHC protocol suitable for use with Protein Block ab64226:
For frozen sections, skip steps 1 and 2. For fluorescent IHC, skip step 3, incubate with fluorescent dye conjugated secondary at step 6, skip rest of steps, and mount with anti-fade mounting medium.1. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section.
2. Use appropriate antigen retrieval buffer or enzyme (primary antibody dependent) to treat sections. Wash 3 times in buffer.
3. Add enough hydrogen peroxide blocking solution to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer. If necessary, use avidin biotin blocking.
4. Apply Protein Block ab64226 (or normal serum from same species as secondary antibody) and incubate for at least 30 minutes at room temperature to block nonspecific background staining. Wash once in buffer.
5. Apply primary antibody in antibody diluent and incubate. Wash 4 times in buffer.
6. Incubate with biotinylated secondary antibody (or HRP polymer secondary antibody and skip step 7). Wash 4 times in buffer.
7. Apply streptavidin-HRP and incubate for 10 minutes at room temperature.
8. Rinse 4 times in buffer. Place slide in DAB substrate or AEC substrate and incubate until desired color is achieved. Rinse 4 times in buffer.
9. Add enough drops of hematoxylin to cover the section. Incubate for 1 minute.
10. Rinse 7-8 times in tap water. Add mounting medium to cover the section.
Find complete IHC kits, and reagents for antigen retrieval, blocking, signal amplification, visualization, counterstaining, and mounting in the IHC kits and reagents guide.
Properties
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Form
Liquid -
Storage instructions
Store at +4°C. -
Storage buffer
pH: 7.2
Preservative: 0.1% Sodium azide
Constituents: PBS, 0.5% Casein, 0.5% BSA -
Concentration information loading... -
Reagent notes
ab64226 reduces the handling of animal serums in the laboratory. The need to match species with the secondary antibody is eliminated due to the lack of normal serum in this product. It has been shown to be effective for immunohistochemistry, ELISA, and blot and requires no mixing or diluting. -
Research areas
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Relevance
Protein Block is used with immunolabeling techniques for the reduction of non-specific background staining.
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab64226 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| IHC-P |
Use at an assay dependent dilution.
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| ELISA |
Use at an assay dependent dilution.
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| WB |
Use at an assay dependent dilution.
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| Notes |
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IHC-P
Use at an assay dependent dilution. |
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ELISA
Use at an assay dependent dilution. |
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WB
Use at an assay dependent dilution. |
Images
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Immunocytochemistry - Protein Block (ab64226)Image from Velez et al., Sci Rep., 10(1):16686; doi: 10.1038/s41598-020-73840-4. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/NUCB1/NLP and NUCB2/NESF colocalizes with GH in somatotrophsRepresentative images of immunofluorescence detection of NUCB1 (green), NUCB2 (green) and GH (red) in GH3 cells. Cells were blocked with an antibody blocking buffer (ABB) based in PBS consisting of 3% BSA, 0.05% Triton X-100 and 10% of protein block solution (ab64226). Cells were counterstained with DAPI (blue) and the images were acquired at 40X magnification.
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Flow Cytometry - Protein Block (ab64226)Image from Lau, et al., PLoS ONE 15, (15(12): e0243506; doi: 10.1371/journal.pone.0243506. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/GLUT2 expression in beta cells of islets after treatment with necrostatin-1.islet were dissociated into single cells, fixed and permeabilised before incubation with Protein Block (ab64226) on ice for 30 minutes to decrease non-specific binding. Then cells were labelled using a FITC-conjugated anti-GLUT2. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)Image from Kljucevic et al., Nutrients, 11(8):1890; doi: 10.3390/nu11081890. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/Immunofluorescence staining of MPO and CD68 in rat heart(A,B) MPO (ab9535, diluted at 1:100) immunoreactivity on a cross section of the heart showing the transmural ischemic zone (IZ) of the anterior wall of the left ventricle with subepicardial (PIZ-EPI) and peripheral (PIZ-1) peri-infarct zones; (C) MPO- (red, ab150084, diluted 1:200) and CD68 (ab31630, diluted at 1:250)-immunoreactivity (green, ab150117, dilued 1:200) of the peri-infarct zone showing no co-localisation of fluorescence; (D) MPO-immunoreactivity is found in the cytoplasm of polymorphonuclear cells (white arrows); (E) a layer of cellular profiles, immunoreactive for both the MPO and CD68 detected within the subepicardial myocardium; (F) CD68-immunoreactivity is found in the cytoplasm of mononuclear cells (white arrows). Scale bars: A = 0.5 mm, B = 200 ?m, C = 75 ?m, D,F = 20 ?m, E = 50 ?m. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)Image from Mohamed et al., Sci Rep., 10: 8840; doi.org/10.1038/s41598-020-64050-z. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/Light micrographs of cerebellar sections with immunohistochemistry by anti-caspase 3 antibodies.Adult male albino rat demonstrating mild caspase-3 immunoreactivity in the three cerebellar layers. Incubation of sections was done with rabbit monoclonal caspase-3 antibodies (Abcam, Cambridge, UK), 1:200 dilutions for 1?h. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)ab64226 was used as pasr of the ab93677 anti-Mouse and Rabbit specific HRP (ABC) Detection IHC kit to perform immunohistochemical analysis of Human Tonsil tissue labeling Ki-67.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)Yu S.H., PLoS One 9 (12), Fig 7c. doi: 10.1371/journal.pone.0114649 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/ab64226 was used in the mouse and rabbit specific HRP/DAB detection IHC Kit (ab64264) to visualize Immunohistochemical analysis staining CD163 in mouse soleus muscle section. Samples were incubated in rabbit CD163 antibody at 1:450 dilution.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (32)
ab64226 has been referenced in 32 publications.
- Lozic M et al. Alteration of Cx37, Cx40, Cx43, Cx45, Panx1, and Renin Expression Patterns in Postnatal Kidneys of Dab1-/- (yotari) Mice. Int J Mol Sci 22:N/A (2021). PubMed: 33525532
- Solic I et al. Expression Pattern of a-Tubulin, Inversin and Its Target Dishevelled-1 and Morphology of Primary Cilia in Normal Human Kidney Development and Diseases. Int J Mol Sci 22:N/A (2021). PubMed: 33800671
- Masoumi KC et al. Integrin a10-Antibodies Reduce Glioblastoma Tumor Growth and Cell Migration. Cancers (Basel) 13:N/A (2021). PubMed: 33803359
- Duplancic R & Kero D Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images. Sci Rep 11:8619 (2021). PubMed: 33883639
- Rodriguez S et al. Characterization of chelator-mediated recovery of pancreatic islets from barium-stabilized alginate microcapsules. Xenotransplantation 27:e12554 (2020). PubMed: 31495985