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FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a

Application

IF, FC, IHC

Reactivity

Mouse

Conjugate

Biotin

Cat no : KFA047

Validation Data Gallery

Applications:
  • Immunofluorescence of HeLa: PFA-fixed HeLa cells were stained with anti-GNL3 antibody (67169-1-lg) labeled with  FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-ATTO594​ (yellow). Nuclei are stained with DAPI (blue). Confocal images were acquired with a 100x oil objective and post-processed. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

    Immunofluorescence of HeLa: PFA-fixed HeLa cells were stained with anti-GNL3 antibody (67169-1-lg) labeled with  FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-ATTO594​ (yellow). Nuclei are stained with DAPI (blue). Confocal images were acquired with a 100x oil objective and post-processed. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

  • Immunofluorescence of rat brain: FFPE rat brain sections were stained with anti-GFAP antibody (60190-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-650 (magenta).  Cell nuclei are in blue.

    Immunofluorescence of rat brain: FFPE rat brain sections were stained with anti-GFAP antibody (60190-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-650 (magenta).  Cell nuclei are in blue.

  • Immunofluorescence of mouse cerebellum: FFPE mouse cerebellum sections were stained with anti-GFAP antibody (60190-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-488 (green),  and anti-SOX10 antibody (66786-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-594 (red). Cell nuclei are stained with DAPI (blue).

    Immunofluorescence of mouse cerebellum: FFPE mouse cerebellum sections were stained with anti-GFAP antibody (60190-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-488 (green),  and anti-SOX10 antibody (66786-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-594 (red). Cell nuclei are stained with DAPI (blue).

  • Flow cytometry with PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD3 antibody (65133-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and PE-Streptavidin. Cells were co-stained with anti-human CD4 antibody (CL488-65143). Cells are not fixed, lymphocytes are gated. 

    Flow cytometry with PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD3 antibody (65133-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and PE-Streptavidin. Cells were co-stained with anti-human CD4 antibody (CL488-65143). Cells are not fixed, lymphocytes are gated. 

  • Flow cytometry with PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD3 antibody (65133-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and PE-Streptavidin. Cells were co-stained with anti-human CD4 antibody (CL488-65143). Cells are not fixed, lymphocytes are gated. 

    Flow cytometry with PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD3 antibody (65133-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and PE-Streptavidin. Cells were co-stained with anti-human CD4 antibody (CL488-65143). Cells are not fixed, lymphocytes are gated. 

  • Flow cytometry of PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD8 antibody (65135-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-PE. Cells were co-stained with anti-human CD3 antibody (CL488-65151). Cells are not fixed, lymphocytes are gated.

    Flow cytometry of PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD8 antibody (65135-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-PE. Cells were co-stained with anti-human CD3 antibody (CL488-65151). Cells are not fixed, lymphocytes are gated.

  • Flow cytometry of PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD8 antibody (65135-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-PE. Cells were co-stained with anti-human CD3 antibody (CL488-65151). Cells are not fixed, lymphocytes are gated.

    Flow cytometry of PBMC. 1X10^6 human PBMC were stained with either mouse IgG2a isotype control or anti-human CD8 antibody (65135-1-Ig), which are labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and Streptavidin-PE. Cells were co-stained with anti-human CD3 antibody (CL488-65151). Cells are not fixed, lymphocytes are gated.

  • Immunohistochemical analysis of paraffin-embedded  human lung cancer tissue slide using anti-CK7 antibody (66483-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and used at a dilution of 1:800 (under 10x lens). Streptavidin Poly-HRP and DAB substrate was used for detection. Heat mediated antigen retrieval performed with Tris-EDTA buffer (pH 9.0).

    Immunohistochemical analysis of paraffin-embedded  human lung cancer tissue slide using anti-CK7 antibody (66483-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and used at a dilution of 1:800 (under 10x lens). Streptavidin Poly-HRP and DAB substrate was used for detection. Heat mediated antigen retrieval performed with Tris-EDTA buffer (pH 9.0).

  • Immunohistochemical analysis of paraffin-embedded  human lung cancer tissue slide using anti-CK7 antibody (66483-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and used at a dilution of 1:800 (under 40x lens). Streptavidin Poly-HRP and DAB substrate was used for detection. Heat mediated antigen retrieval performed with Tris-EDTA buffer (pH 9.0).

    Immunohistochemical analysis of paraffin-embedded  human lung cancer tissue slide using anti-CK7 antibody (66483-1-Ig) labeled with FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a (KFA047) and used at a dilution of 1:800 (under 40x lens). Streptavidin Poly-HRP and DAB substrate was used for detection. Heat mediated antigen retrieval performed with Tris-EDTA buffer (pH 9.0).

Product Information


Product name                

FlexAble Biotin Antibody Labeling Kit for Mouse IgG2a                

Assay type                

Antibody labeling                

Tested applications                

IF, FC, IHC               

Species Reactivity                

Mouse IgG2a                

Antibody amount per labeling reaction            

0.5 µg antibody                               

Conjugate                

Biotin               

Compatible with               

Streptavidin conjugated dyes and molecules 



Component            10 rxns            50 rxns
           		4×50 rxns            
Biotin FlexLinker for Mouse IgG2a10 μL50 μL4×50 μL
FlexQuencher for Mouse IgG2a20 μL100 μL4×100 μL
FlexBuffer100 μL500 μL4×500 μL



Storage

Store for 1 year at -20°C or for 6 months at +4°C after shipment. Avoid exposure to light.

Others

Q: What are the FlexLinker, FlexQuencher and FlexBuffer?

A: The FlexLinker is a small polypeptide to which dyes are covalently conjugated that can label unconjugated primary antibodies. The FlexQuencher is an Fc-containing fragment that neutralizes the excess FlexLinker. The FlexBuffer is a PBS-based buffer.


Q: What is the largest quantity I can label?

A: With a standard kit size (50 reactions), you can label 25 µg of one antibody or up to 50 different antibodies. You can easily scale up the antibody amount per labeling approach.


Q: What is the lowest concentration of my primary antibody that I can use?

A: Our protocol uses 0.5 µg of primary antibody in 7 µL, which ends up at 0.07 mg/mL. If the concentration of your antibody is lower, you can also use a larger volume than 7 µL.


Q: Can I label primary antibodies stored in BSA, glycerol, Tris buffer and/or preservatives?

A: Yes, FlexAble Antibody Labeling Kits have been validated with carriers and amine buffers. Neither BSA nor amine buffers, in any chosen concentration, interfere with the labeling. 50% glycerol as well as preservatives like sodium azide are also compatible with the kit.


Q: How many different primary antibodies can I label with one kit?

A: You can label up to 50 different antibodies with our FlexAble 50 rxn Kit, and up to 10 antibodies with our FlexAble 10 rxn Kit.


Q: Will I observe cross-reactivity/leaking when I use two FlexAble-labeled antibodies from the same species during multiplexing?

A: FlexAble labels primary antibodies with a high affinity FlexLinker. Dissociation of FlexLinker from one antibody and association to another antibody is rare. If you observe leaking, we recommend adding more FlexQuencher to remove unbound FlexLinker, or you can try sequential staining of the labeled antibodies. 


More FAQs