Anti-TRK fused gene antibody [EPR8766]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRK fused gene antibody [EPR8766] (AB156866)

Immunohistochemical analysis of paraffin embedded Human thyroid carcinoma tissue labeling TRF fused gene with ab156866 at 1/50.

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRK fused gene antibody [EPR8766] (AB156866)

Immunohistochemical analysis of paraffin embedded Human colon tissue labeling TRF fused gene with ab156966 at 1/50.

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TRK fused gene antibody [EPR8766] (AB156866)

Immunocytochemistry/Immunofluorescence analysis of MCF-7 (human breast carcinoma) cells labelling TRK fused gene with purified ab156866 at 1/1000. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue).

Secondary Only Control : PBS was used instead of the primary antibody as the negative control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TRK fused gene antibody [EPR8766] (AB156866)

Immunofluorescent analysis of HeLa cells labeling TRF fused gene with ab156866 at 1/100.

• IP

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Immunoprecipitation - Anti-TRK fused gene antibody [EPR8766] (AB156866)

Immunoprecipitaion analysis in 293T cell lysate of TRK fused gene immunoprecipitated with ab156866 at 1/10.

All lanes:

Immunoprecipitation - Anti-TRK fused gene antibody [EPR8766] (ab156866) at 1/10000 dilution

All lanes:

Immunoprecipitation pellets from 293T cell lysate at 10 µg

Predicted band size: 35 kDa,43 kDa

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• WB

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Western blot - Anti-TRK fused gene antibody [EPR8766] (AB156866)

Lanes 1-4 : Merged signal (red and green). Green - ab156866 observed at 57 kDa. Red - loading control ab8245 observed at 36 kDa.

ab156866 Anti-TRK fused gene antibody [EPR8766] was shown to specifically react with TFG in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265841 (knockout cell lysate ab257738) was used. Wild-type and TFG knockout samples were subjected to SDS-PAGE. ab156866 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-TRK fused gene antibody [EPR8766] (ab156866) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

TFG knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human TFG (TRK fused gene) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-tfg-trk-fused-gene-knockout-hela-cell-line-ab265841'>ab265841</a>)

Lane 3:

HAP-1 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 43 kDa

Observed band size: 57 kDa

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• WB

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Western blot - Anti-TRK fused gene antibody [EPR8766] (AB156866)

All lanes:

Western blot - Anti-TRK fused gene antibody [EPR8766] (ab156866) at 1/10000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

A549 cell lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Predicted band size: 43 kDa

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• WB

Lab

Western blot - Anti-TRK fused gene antibody [EPR8766] (AB156866)

Lanes 1 - 4 : Merged signal (red and green). Green - ab156866 observed at 57 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab156866 was shown to react with TFG in HAP1 wild-type cells in Western blot. Loss of signal was observed when TFG knockout sample was used. HAP1 wild-type and TFG knockout whole cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab156866 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-TRK fused gene antibody [EPR8766] (ab156866) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

TFG knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 57 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-TRK fused gene antibody [EPR8766] (AB156866)

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.