Anti-APOBEC3B antibody [EPR18138]

• WB

Lab

Western blot - Anti-APOBEC3B antibody [EPR18138] (AB184990)

Negative sample : SK-BR-3 (PMID : 27634334).

Merged signal (red and green).
Green - ab184990 observed at 38 kDa.
Red - loading control, ab8245 observed at 36 kDa.

ab184990 Anti-APOBEC3B antibody [EPR18138] was shown to specifically react with APOBEC3B in wild-type HeLa cells. Loss of signal was observed when APOBEC3B CRISPR-Edited HeLa cell line was used. Wild-type and APOBEC3B CRISPR-Edited HeLa samples were subjected to SDS-PAGE.

ab184990 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-APOBEC3B antibody [EPR18138] (ab184990) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

APOBEC3B CRISPR-Edited HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 3:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

SK-BR-3 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

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

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Observed band size: 38 kDa

false

• WB

Lab

Western blot - Anti-APOBEC3B antibody [EPR18138] (AB184990)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM /TBST We are unsure of the identity of the 55kda band.

All lanes:

Western blot - Anti-APOBEC3B antibody [EPR18138] (ab184990) at 1/500 dilution

All lanes:

A431 (Human epidermoid carcinoma epithelial cell) 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: 46 kDa

Observed band size: 38 kDa

false

Exposure time: 180s

• WB

Collaborator

Western blot - Anti-APOBEC3B antibody [EPR18138] (AB184990)

Blocking buffer : 5% NFDM/TBST.

Dilution buffer : 1% NFDM/TBST.

Exposure time : Panel A : 20 s; Panel B : 1 min.

Figure provided by Dr. Jenni Nikkila. Gene Function Lab at The Institute of Cancer Research, London. HEK293-APOBEC3B(+GFP) tetracycline-inducible cell line provide by Dr Reuben Harris, University of Minnesota, Minneapolis - cited in Akre, M.K, G.J. Starrett, J.S. Quist, N.A. Temiz, M.A. Carpenter, A.N.J. Tutt, A. Grigoriadis & R.S. Harris (2016) Mutation processes in 293-based clones overexpressing the DNA cytosine deaminase APOBEC3B. PLoS One11(5) : e0155391. doi : 10.1371/journal.pone.0155391.

The 35 kDa band represents a known splice variant of APOBEC3B.

All lanes:

Western blot - Anti-APOBEC3B antibody [EPR18138] (ab184990) at 1/10000 dilution

Lane 1:

HepaRG-APOBEC3B tetracycline-inducible cell line (+/-) at 20 µg

Lane 2:

HCC1143 cell line infected with control (+) and APOBEC3B lentivirus (-) at 20 µg

Lane 3:

HEK293-APOBEC3B(+GFP) tetracycline-inducible cell line (+/-) at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (H+L) conjugated to HRP at 1/10000 dilution

Predicted band size: 46 kDa

Observed band size: 35 kDa,70 kDa

false

• WB

Lab

Western blot - Anti-APOBEC3B antibody [EPR18138] (AB184990)

Exposure time : Lane 1-3 : 20 seconds; Lane 4-5 : 180 seconds

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

ab184990 only detects very weak signal in HepG2 and HeLa cell lines.

All lanes:

Western blot - Anti-APOBEC3B antibody [EPR18138] (ab184990) at 1/1000 dilution

Lane 1:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 4:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 3 and 5:

HeLa (Human epithelial cell line from cervix adenocarcinoma) 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

Observed band size: 38 kDa

false

• WB

Supplier Data

Western blot - Anti-APOBEC3B antibody [EPR18138] (AB184990)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-APOBEC3B antibody [EPR18138] (ab184990) at 1/1000 dilution

Lane 1:

Human APOBEC3B fragment recombinant protein at 0.01 µg

Lane 2:

Human APOBEC3G fragment recombinant protein at 0.01 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 1/1000 dilution

Predicted band size: 46 kDa

Observed band size: 46 kDa

false