Anti-Dysferlin antibody [JAI-1-49-3]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue labelling Dysferlin with purified ab124684 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Immunocytochemistry/Immunofluorescence analysis of A673 cells labelling Dysferlin with purified ab124684 at 1/300. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/300) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue labelling Dysferlin with unpurified ab124684 at a dilution of 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Immunohistochemistry (Frozen sections) analysis of unfixed frozen human skeletal muscle tissue (control) labelling Dysferlin with unpurified ab124684 at a dilution of 1/200.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Immunohistochemistry (Frozen sections) analysis of unfixed frozen mouse skeletal muscle tissue (wild type) labelling Dysferlin with unpurified ab124684 at a dilution of 1/200.

• WB

Lab

Western blot - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Dysferlin antibody [JAI-1-49-3] (ab124684) at 1/1000 dilution

All lanes:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 237 kDa

Observed band size: 280 kDa

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• WB

Lab

Western blot - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Dysferlin antibody [JAI-1-49-3] (ab124684) at 1/1000 dilution

All lanes:

Mouse muscle tissue lysate at 10 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 237 kDa

Observed band size: 280 kDa

false

• WB

Unknown

Western blot - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

All lanes:

Western blot - Anti-Dysferlin antibody [JAI-1-49-3] (ab124684) at 1/1000 dilution

Lane 1:

Human skeletal muscle tissue lysate (control) at 10 µg

Lane 2:

Human skeletal muscle tissue lysate (LGMD2B) at 10 µg

Lane 3:

Mouse skeletal muscle tissue lysate (wild-type mice) at 10 µg

Lane 4:

Mouse skeletal muscle tissue lysate (Dysf-/- transgenic mouse) at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 237 kDa

Observed band size: 280 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-Dysferlin antibody [JAI-1-49-3] (AB124684)

Dysferlin western blot using anti-Dysferlin antibody [JAI-1-49-3] ab124684. Publication image and figure legend from Vanhoutte, D., Schips, T. G., et al., 2016, Elife, PubMed 27669143.

ab124684 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab124684 please see the product overview.

Thbs4 enhances stabilizing proteins at the sarcolemma.(A) Immunofluoresence (green) detection of δ-, α-, β-, and γ-sarcoclycan (SGC), β-dystroglycan (β-DG), utrophin (Utro.), dystrophin (Dystro.) and β1D-integrin in littermates of three month-old WT, Thbs4-Tg, Sgcd-/- and Sgcd-/-Thbs4-Tg quadriceps. Representative images of 4 mice per genotype are shown. Scale bar = 25 μm. (B) Representative Western blots of sarcolemmal protein extracts (upper) or total cytoplasmic protein extracts (lower) from the quadriceps of the indicated groups of mice for the indicated proteins (n = 4–5 biological replicates). Abbreviations : Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5- integrin. The red boxes show increased protein levels. Also see Figure 6—figure supplement 1 for replicates. (C) Representative immunoblot for structural components of the DGC- and integrin-associated protein complexes in sarcolemmal preparations from Thbs4-/- and WT quadriceps at four months of age (n = 4 biological replicates). The burgundy-boxed areas show reduced protein levels. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls for sarcolemmal protein extracts; Gapdh was used as loading control for total cell protein extracts.DOI : http : //dx.doi.org/10.7554/eLife.17589.018Thbs4 enhances stabilizing proteins at the sarcolemma and directly interacts with integrins.(A,B) Representative Western blots of sarcolemmal protein extracts from the quadriceps of the indicated groups of mice at three months of age (n = 4–5 biological replicates). Sgcd-/- Tg and mdx Tg indicate Sgcd-/- and mdx with skeletal muscle specific Thbs4 overexpression, respectively. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls. Abbreviations : Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-itg (integrin). (C) Immunoblots for β1D- and α7-Integrin (Itg), β-dystroglycan (DG) and Thbs4 (Flag) from neonatal rat ventricular myocyte extracts immunoprecipitated with a Flag antibody (Thbs4). Adβgal was used as a control infection (n = 3 biological replicates). An adenovirus expressing a Flag-tagged Thbs4 protein was used to achieve high level of this protein to identify the interaction. (D,E) Representative Western blots for Thbs4, α5- and β1D-integrin (itg) from intracellular vesicular isolates from WT and Thbs4 Tg quadriceps (D) or WT and Sgcd-/-quadriceps (E) that were immunoprecipitated with an antibody raised against the cytoplasmic domain of β1D-integrin (n = 3 biological replicates), showing that Thbs4 and α5-integrin localize to β1D-integrin-positive intracellular vesicles. α-tubulin and Gapdh are presented as loading control.DOI : http : //dx.doi.org/10.7554/eLife.17589.019

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