Anti-ALKBH1 antibody [EPR6176]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH1 antibody [EPR6176] (AB126596)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling ALKBH1 with purified ab126596 at a dilution of 1/50. Heat mediated antigen retrieval was performed using 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH1 antibody [EPR6176] (AB126596)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling ALKBH1 with unpurified ab126596 at a dilution of 1/50.

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

• IP

Lab

Immunoprecipitation - Anti-ALKBH1 antibody [EPR6176] (AB126596)

ab126596 (purified) at a dilution of 1/30 immunoprecipitating ALKBH1 in Jurkat whole cell lysate.

Lane 1 (input) : Jurkat whole cell lysate (10μg)

Lane 2 (+) : ab126596 + Jurkat whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab126596 in Jurkat whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-ALKBH1 antibody [EPR6176] (ab126596)

Predicted band size: 43 kDa

Observed band size: 43 kDa

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• IP

Lab

Immunoprecipitation - Anti-ALKBH1 antibody [EPR6176] (AB126596)

ab126596 (purified) at a dilution of 1/30 immunoprecipitating ALKBH1 in K562 whole cell lysate.

Lane 1 (input) : K562 whole cell lysate (10μg)

Lane 2 (+) : ab126596 + K562 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab126596 in K562 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-ALKBH1 antibody [EPR6176] (ab126596)

Predicted band size: 43 kDa

Observed band size: 43 kDa

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• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR6176] (AB126596)

Lanes 1 - 4 : Merged signal (red and green). Green - ab126596 observed at 43 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab126596 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% Milk. ab126596 and ab8245 (Mouse anti GAPDH 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 [EPR6176] (ab126596) 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

Observed band size: 43 kDa

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• WB

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Western blot - Anti-ALKBH1 antibody [EPR6176] (AB126596)

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR6176] (ab126596) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

K562 cell lysate at 10 µg

Lane 3:

A549 cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 43 kDa

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• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR6176] (AB126596)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR6176] (ab126596) at 1/10000 dilution

Lane 1:

Jurkat whole cell lysate at 20 µg

Lane 2:

K562 whole cell lysate at 20 µg

Lane 3:

A549 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

Predicted band size: 35 kDa,43 kDa,58 kDa,61 kDa

Observed band size: 36 kDa,40 kDa,40-45 kDa,43 kDa,61 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-ALKBH1 antibody [EPR6176] (AB126596)

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-ALKBH1 antibody [EPR6176] (AB126596)

ALKBH1 western blot using anti-ALKBH1 antibody [EPR6176] ab126596. Publication image and figure legend from Guo, Y., Pei, Y., et al., 2020, Aging (Albany NY), PubMed 32283543.

ab126596 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 ab126596 please see the product overview.

Elevated ALKBH1 level reduced 6mA DNA level in human aortic smooth muscle cells (HASMCs). (A–C) Representative western blot and quantification of N6AMT1 and ALKBH1 protein levels in HASMCs treated with various concentrations of angiotensin II (Ang II) or endothelin-1 (ET1) for 72 h. GAPDH was the internal control. (D, E) Global 6mA level measured by ELISA assay in Ang II- or ET1–treated HASMCs. (F, G) ALKBH1 mRNA level by RT-qPCR assay (F) and global m6A level by ELISA analysis (G) in Ang II- or ET1–treated HASMCs with siRNA-Control (Si-CN) or siRNA-ALKBH1 transfection. Data are mean ± SD (n= 4/group) and were analyzed by one-way ANOVA, followed by Bonferroni's multiple comparison test. *p<0.05, **p<0.01 vs the control of Ang II or ET-1. #p<0.05 vs Si-CN and Ang II or ET1 treatment for F and G.

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