Anti-ALKBH1 antibody [EPR6175(2)]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH1 antibody [EPR6175(2)] (AB128895)

ab128895, at a dilution of 1/50, staining ALKBH1 in paraffin-embedded Human breast carcinoma tissue by Immunohistochemistry.

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

• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR6175(2)] (AB128895)

Lanes 1 - 4 : Merged signal (red and green). Green - ab128895 observed at 43 kDa. Red - loading control, ab7291, observed at 50 kDa.

ab128895 was shown to specifically react with in wild-type HAP1 cells as signal was lost in ALKBH1 knockout cells. Wild-type and ALKBH1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. ab128895 and ab7291 (Mouse anti Tubulin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR6175(2)] (ab128895) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

ALKBH1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

K562 whole cell lysate at 20 µg

Lane 4:

A549 whole cell lysate at 20 µg

Predicted band size: 43 kDa

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• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR6175(2)] (AB128895)

Blocking and diluting buffer : 5% NFDM /TBST

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR6175(2)] (ab128895) at 1/1000 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) transfected with siRNA specifically targeting ALKBH1 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 120s

• WB

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Western blot - Anti-ALKBH1 antibody [EPR6175(2)] (AB128895)

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR6175(2)] (ab128895) at 1/1000 dilution

Lane 1:

A431 cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

A549 cell lysate at 10 µg

Lane 4:

K562 cell lysate at 10 µg

Lane 5:

Raw 264.7 cell lysate at 10 µg

Lane 6:

NIH3T3 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-ALKBH1 antibody [EPR6175(2)] (AB128895)

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.