F(ab) and F(ab')2 fragment secondary antibodies are antigen-binding fragments that are derived from full-length secondary antibodies, conventionally through enzymatic cleavage. These fragments offer several advantages over whole IgG antibodies due to their smaller size and the absence of the Fc region.

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What are F(ab) and F(ab’)2 fragment antibodies?

F(ab) fragment antibodies are obtained by papain cleavage of whole IgG antibodies, resulting in the removal of the Fc region and hinge region. This makes them monovalent in nature and about one third the size of whole IgG antibodies (~50 kDa).

F(ab')2 fragment antibodies, on the other hand, are obtained by pepsin cleavage of whole IgG antibodies. They lack the Fc region but retain the hinge region, which holds the two antigen binding sites together through disulfide bonds. This makes F(ab')2 fragments bivalent and about two thirds the size of whole IgG antibodies (~100 kDa).

Both F(ab) and F(ab')2 fragment antibodies can be conjugated with various labels, such as fluorophores or enzymes, for detection and visualization in immunoassays. These fragments are widely used in research to enhance specificity and sensitivity, helping lead to accurate and reliable experimental results. Their versatility makes them valuable research tools.

Learn more about Antibody Fragmentation

Illustration of F(ab) fragment in comparison to whole IgG
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Figure 1. Depiction of F(ab) fragment antibodies. This figure depicts that a F(ab) fragment derived from the full-length antibody consists of a single copy of the VH, CH, VL and CL domains, that together form a monovalent antigen binding fragment.
Illustration of F(ab’)2 fragment in comparison to whole IgG
 Click image to enlarge
Figure 2. Depiction of F(ab’)2 fragment antibodies. This figure depicts that a F(ab’)2 fragment derived from the full-length antibody consists of two copies of the VH, CH, VL and CL domains held together by disulfide bonds between the hinge peptides extending from the two CH domains. Together, this forms a bivalent antigen binding fragment.

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Alexa Fluor conjugated F(ab) antibodies


Benefits of antibody fragments

F(ab) antibody fragments offer several benefits over whole IgG antibodies due to their smaller size and the absence of the Fc region. The smaller size enables better penetration into tissue sections and improved staining in applications such as immunohistochemistry (IHC). The lack of the Fc region results in reduced nonspecific background when staining cells and tissues with Fc receptors, such as immune cells, and other Fc interaction-related interference. The lack of the Fc region can also reduce the immunogenicity of F(ab) and F(ab’)2 fragments compared to whole IgG antibodies. Fc-specific F(ab) fragments also offer expanded multiplexing options beyond what is achievable with whole IgG antibodies for applications such as immunocytochemistry, immunohistochemistry, flow cytometry, and western blotting. For example, it is possible to use a F(ab) fragment goat anti-rabbit Fc-specific secondary antibody along with F(ab) fragment rabbit anti-goat Fc-specific secondary antibody in the same experiment. However, using a full-length goat anti-rabbit secondary antibody along with a full-length rabbit anti-goat secondary antibody would result in poor staining as the two secondary antibodies tend to bind each other rather than the target primary antibody.

While F(ab) fragments are monovalent and smaller, offering advantages in terms of reduced immunogenicity and sequential labeling of multiple primary antibodies from the same host, F(ab')2 fragments may provide higher avidity and improved target capture. Understanding the properties and advantages of these fragments allow for better experimental design to achieve improved sensitivity and specificity.

Comparison of antibody formats:

PropertyF(ab) fragmentF(ab’)2 fragmentWhole IgG
Fc-FcR based nonspecific interaction-++++
Tissue penetration ability++++++
Multiplexing abilities (using Fc specific secondary antibodies)+++++
Sequential labeling of multiple primaries of the same species/isotype+--
Immunogenicity++++++
ValencyMonovalentBivalentBivalent
Signal amplification++++++

- indicates the specified property does not apply to the antibody format
+ indicates the antibody format presents the specified property
++ indicates the antibody format presents the specified property to a greater extent
+++ indicates the antibody format presents the specified property to the greatest extent

Learn more about advantages of antibody fragments here


Search F(ab) and F(ab’)2 fragment secondary antibodies by application

Figure 3. Immunofluorescence analysis to compare multiplexing capabilities of Fc-specific F(ab) fragment secondary antibodies versus whole IgG secondary antibodies. Immunofluorescence analysis was performed on fixed and permeabilized MCF7 cells blocked with 2% BSA at room temperature and stained with a cocktail of H3K4me3 Recombinant Rabbit Monoclonal Antibody (14H24L5) (Cat. No. 703849; 1:100 dilution), beta Tubulin Monoclonal Antibody (2 28 33) (Cat. No. 32-2600; 1:500 dilution), and TROP2 Polyclonal Antibody (Cat. No. PA5-47030; 1:100 dilution) in 0.1% BSA overnight at 4-degree Celsius. This was detected by incubating with a cocktail of F(ab)-Goat anti-Rabbit IgG Fc Secondary Antibody, Alexa Fluor 488 (Cat. No. A66785; used at 1 µg/mL), F(ab)-Goat anti-Mouse IgG1 Fc Secondary Antibody, Alexa Fluor 555 (Cat. No. A66782; used at 1 µg/mL), and F(ab)-Rabbit anti-Goat IgG Fc Secondary Antibody, Alexa Fluor 647 (Cat. No. A66784; used at 1 µg/mL) or a cocktail of Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Cat. No. A11008; used at 4 µg/mL), Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Cat. No. A21422; used at 2 µg/mL), and Rabbit Anti-Goat IgG H&L, Alexa Fluor 647 for 1 hour at room temperature. Panels a-e represent images from sample stained with Fc-specific F(ab) secondaries. Panel a) shows cells stained with the nuclear stain, Hoechst 33342 (Cat. No. H1399; 1:500 dilution) Panel b) shows the nuclear expression of H3K4me3. Panel c) shows the cytosolic expression of beta-tubulin protein. Panel d) shows the plasma membrane expression of TROP2 protein. Panel e) shows cells stained with Alexa Fluor Plus 750 Phalloidin (Cat. No. A30105; 1:400 dilution). Panels f-j represent images from sample stained with full length IgG secondaries. Panel f) shows cells stained with the nuclear stain, Hoechst 33342 (Cat. No. H1399; 1:500 dilution) Panel g) shows the lack of nuclear expression of H3K4me3. Panel h) shows the cytosolic expression of beta-tubulin protein. Panel i) shows the lack of plasma membrane expression of TROP2 protein. Panel j) shows cells stained with Alexa Fluor Plus 750 Phalloidin (Cat. No. A30105; 1:400 dilution). The lack of signal for detection of H3K4me3 and TROP2 is due to the incompatibility in using Goat anti-Rabbit and Rabbit anti-Goat full-length IgG secondary antibodies in the same experiment. The images were captured at 40X magnification on CellInsight CX7 LZR High-Content Screening (HCS) Platform (Cat. No. CX7A1110LZR).

Figure 4. Stained HeLa cells. Immunofluorescence analysis of F(ab)-Goat anti-Mouse IgG1 Fc Secondary Antibody, Alexa Fluor 568 (Cat. No. A66783) was performed on fixed and permeabilized HeLa cells blocked with 2% BSA at room temperature and stained with beta Tubulin Monoclonal Antibody (1 µg/mL; Cat. No. 32-2600) in 0.1% BSA overnight at 4°C. This was detected by incubating with F(ab)-Goat anti-Mouse IgG1 Fc Secondary Antibody, Alexa Fluor 568 (1 µg/mL; Cat. No. A66783) for 1 hour at room temperature. Figure shows the composite image of cells stained for detection of beta tubulin protein and the nucleus stained with Hoechst 33342 (1:500; Cat. No. H1399). Images were captured at 20X magnification on CellInsight CX7 LZR High-Content Screening (HCS) Platform (Cat. No. CX7A1110LZR).

Figure 5. Stained mouse colon cells. Immunohistochemical analysis of F(ab)-Goat anti-Mouse IgG1 Fc Secondary Antibody, Alexa Fluor 488 (Cat. No. A66781) was performed on mouse colon FFPE sections. De-paraffinized sections were subjected to heat induced epitope retrieval using eBioscience IHC Antigen Retrieval Solution - High pH (10X) (Cat. No. 00-4956-58) and blocked with 2% normal goat serum for 1 hour at room temperature. Sections were then probed with Desmin Monoclonal Antibody (DE-U-10), eBioscience (5 µg/mL; Cat. No. 14-9747-82) overnight at 4°C. This was detected by incubating with F(ab)-Goat anti-Mouse IgG1 Fc Secondary Antibody, Alexa Fluor 488 (1 µg/mL; Cat. No. A66781) in 0.1% normal goat serum for 45 minutes at room temperature. Autofluorescence was quenched using ReadyProbes Tissue Autofluorescence Quenching Kit (Cat. No. R37630) and nuclei were stained with DAPI (Cat. No. D1306). Sections were mounted using ProLong Glass Antifade Mountant (Cat. No. P36984). The images were captured on EVOS M7000 Imaging System (Cat. No. AMF7000) at 20X magnification. Figure shows the composite image of colon stained for Desmin protein and nuclear stain.

Figure 6. Stained 769P cells. 769P cells were stained with 1 µg/mL of Rabbit IgG Isotype Control (RbNP15), eBioscience (Cat. No. 14-4616-82) (blue histogram) or CD365 (Tim-1) Recombinant Rabbit Monoclonal Antibody (P365D), eBioscience (Cat. No. 14-3658-82) (pink histogram) followed by F(ab)-Goat anti-Rabbit IgG Fc Secondary Antibody, Alexa Fluor 647 (Cat. No. A66788) at a concentration of 1 µg/mL. Viable cells were used for flow cytometry analysis, as determined by LIVE/DEAD Fixable Green Dead Cell Stain Kit, for 488 nm excitation (Cat. No. L23101).

Figure 7. Western blot analysis of F(ab) fragments. Western blot analysis of F(ab)-Goat anti-Mouse IgG1 Fc Secondary Antibody, Alexa Fluor 647 (Cat. No. A66784) was performed by loading 64 µg (Lane 1), 32 µg (Lane 2), 16 µg (Lane 3) and 8 µg (Lane 4) per well of HeLa whole cell extracts and were electrophoresed using Novex NuPAGE 4-12 % Bis-Tris gel (Cat. No. NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Cat. No. IB23001) by iBlot 2 Dry Blotting System (Cat. No. IB21001). The blot was probed with beta Tubulin Monoclonal Antibody (2 28 33) (Cat. No. 32-2600) at a dilution of 1:5,000 and detected by fluorescent imaging with F(ab)-Goat anti-Mouse IgG1 Fc Secondary Antibody, Alexa Fluor 647 (Cat. No. A66784) at a dilution of 1:500 using the iBright FL 1500 (Cat. No. A44115). This antibody can be used in multiplex fluorescent western blotting experiments as demonstrated in the figure using HSP90 alpha Polyclonal Antibody (Cat. No. PA3-013) at a dilution of 1:5,000 and its respective conjugated secondary antibody F(ab)-Goat anti-Rabbit IgG Fc Secondary Antibody, Alexa Fluor 488 (Cat. No. A66785) at a dilution of 1:500.

Figure 8. Alexa Fluor Plus 488 dye-stained human colon. Human colon FFPE tissue sections were deparaffinized through a series of xylene and graded ethanol washes with a final wash in ddH2O. Antigen retrieval was performed by pressure cooking tissue in IHC Antigen Retrieval Solution - Low pH (Cat. No. 00-4955-58) for 10 minutes followed by a slow, 20 minute cool down. Slides were dried and barriers marked with a PAP Pen. Tissues were permeabilized with 0.5% Triton X-100 in PBS for 15 minutes and removed. Tissues were blocked with 3% BSA/5% normal goat serum/PBS. Following removal of blocking solution, tissues were treated with 300 µL of Mouse monoclonal anti-Cadherin-17 (Cat. No. MA5-29135) diluted 1:250 in blocking solution overnight at 4°C in a humid chamber. Tissues were washed 3 x 5 minutes in 225 mL PBS. 300 µL of F(ab’)2 Goat anti-Mouse IgG Alexa Fluor Plus 488 (green, Cat. No. A48286) diluted 1:500 in blocking solution was added to tissue and incubated for 1 hour followed by 3 x 5 minute washes in PBS. Following wash, Hoechst 33342 (blue, Cat. No. H1399) diluted 1:4,000 and Tomato Lectin DyLight 649 (red, Cat. No. L32472) diluted 1:100 in blocking buffer were added to tissue for 30 minutes followed by two quick washes in PBS and one final rinse in ddH2O. Tissues were mounted with ProLong Glass reagent (Cat. No. P36982) and imaged on an EVOS M7000 system (Cat. No. AMF7000).

Figure 9. Alexa Fluor Plus 555 dye-stained human colon. Human colon FFPE tissue sections were deparaffinized through a series of xylene and graded ethanol washes with a final wash in ddH2O. Antigen retrieval was performed by pressure cooking tissue in IHC Antigen Retrieval Solution - Low pH (Cat. No. 00-4955-58) for 10 minutes followed by a slow, 20 minute cool down. Slides were dried and barriers marked with a PAP Pen. Tissues were permeabilized with 0.5% Triton X-100 in PBS for 15 minutes and removed. Tissues were blocked with 3% BSA/5% normal goat serum/PBS. Following removal of blocking solution, tissues were treated with 300 µL of Mouse monoclonal anti-Cadherin-17 (Cat. No. MA5-29135) diluted 1:250 in blocking solution overnight at 4°C in a humid chamber. Tissues were washed 3 x 5 minutes in 225 mL PBS. 300 µL of F(ab’)2 Goat anti-mouse IgG Alexa Fluor Plus 555 (orange, Cat. No. A48287) diluted 1:500 in blocking solution was added to tissue and incubated for 1 hour followed by 3 x 5 minute washes in PBS. Following wash, Hoechst 33342 (blue, Cat. No. H1399) diluted 1:4,000 and Tomato Lectin DyLight 488 (green, Cat. No. L32470) diluted 1:100 in blocking buffer were added to tissue for 30 minutes followed by two quick washes in PBS and one final rinse in ddH2O. Tissues were mounted with ProLong Glass reagent (Cat. No. P36982) and imaged on an EVOS M7000 system (Cat. No. AMF7000).

    

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