Anti-RALBP1 antibody [EPR6472]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RALBP1 antibody [EPR6472] (AB133549)

Overlay histogram showing HEK293 cells stained with ab133549 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab133549, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RALBP1 antibody [EPR6472] (AB133549)

Immunohistochemical analysis of paraffin embedded Human lung tissue labelling RALBP1 with ab133549 antibody at a dilution of 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-RALBP1 antibody [EPR6472] (AB133549)

Lanes 1-3 : Merged signal (red and green). Green - ab133549 observed at 95 kDa. Red - loading control ab8245 observed at 37 kDa.

ab133549 Anti-RALBP1 antibody [EPR6472] was shown to specifically react with PDE10A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265404 (knockout cell lysate ab258167) was used. Wild-type and PDE10A knockout samples were subjected to SDS-PAGE. ab133549 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-RALBP1 antibody [EPR6472] (ab133549) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RALBP1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RALBP1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ralbp1-knockout-hela-cell-line-ab265404'>ab265404</a>)

Lane 3:

Daudi 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 1/20000 dilution

Predicted band size: 76 kDa

Observed band size: 95 kDa

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• WB

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Western blot - Anti-RALBP1 antibody [EPR6472] (AB133549)

All lanes:

Western blot - Anti-RALBP1 antibody [EPR6472] (ab133549) at 1/10000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

HT-29 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 76 kDa

Observed band size: 95 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-RALBP1 antibody [EPR6472] (AB133549)

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.