Anti-Histone H3 (ubiquityl K23) antibody [EPR18876]

• IP

Supplier Data

Immunoprecipitation - Anti-Histone H3 (ubiquityl K23) antibody [EPR18876] (AB194243)

Histone H3 (ubiquityl K23) was immunoprecipitated using ab194243 at 1/30 dilution from 0.35 mg of whole cell lysate from HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 2mM thymidine for 18 hours, followed by addition of fresh culture medium and incubation for 9 hours, then treated with 2mM thymidine for 15 hours, followed by addition of fresh culture medium and incubation for 4 hours.

Western blot was performed from the immunoprecipitate using ab194243 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : 10 μg of whole cell lysate from HeLa treated with 2mM thymidine for 18 hours, followed by addition of fresh culture medium and incubation for 9 hours, cells were then treated with 2mM thymidine for 15 hours, followed by addition of fresh culture medium and incubation for 4 hours (Input).

Lane 2 : ab194243 IP in whole cell lysate from HeLa treated with 2mM thymidine for 18 hours, followed by addition of fresh culture medium and incubation for 9 hours, cells were then treated with 2mM thymidine for 15 hours, followed by addition of fresh culture medium and incubation for 4 hours.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab194243 in whole cell lysate from HeLa treated with 2mM thymidine for 18 hours, followed by addition of fresh culture medium and incubation for 9 hours, cells were then treated with 2mM thymidine for 15 hours, followed by addition of fresh culture medium and incubation for 4 hours.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 seconds.

The band at around 35kDa can be explained as di-ubiquitinated K23 (PMID24013172).

All lanes:

Immunoprecipitation - Anti-Histone H3 (ubiquityl K23) antibody [EPR18876] (ab194243)

Predicted band size: 15 kDa

false

• WB

Supplier Data

Western blot - Anti-Histone H3 (ubiquityl K23) antibody [EPR18876] (AB194243)

Blocking/Dilution buffer : 5% NFDM/TBST.

The MW is consistent with literature : PMID 24013172.

All lanes:

Western blot - Anti-Histone H3 (ubiquityl K23) antibody [EPR18876] (ab194243) at 1/20000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate

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 1/100000 dilution

Predicted band size: 15 kDa

Observed band size: 28 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Histone H3 (ubiquityl K23) antibody [EPR18876] (AB194243)

Blocking/Dilution buffer : 5% NFDM/TBST.

The expression level of H3K23ubi is increased during S phase. PMID 24013172.

The MW is consistent with literature : PMID 24013172.

All lanes:

Western blot - Anti-Histone H3 (ubiquityl K23) antibody [EPR18876] (ab194243) at 1/20000 dilution

Lane 1:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

Whole cell lysate from HeLa treated with 2mM thymidine for 18 hours, followed by addition of fresh culture medium and incubation for 9 hours. Cell were then treated with 2mM thymidine for 15 hours, followed by addition of fresh culture medium and incubation for 4 hours at 10 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 1/100000 dilution

Predicted band size: 15 kDa

Observed band size: 28 kDa

false

Exposure time: 3min

• Dot

Supplier Data

Dot Blot - Anti-Histone H3 (ubiquityl K23) antibody [EPR18876] (AB194243)

Dot blot analysis of Histone H3 (ubiquityl K23) labeled with ab194243 at 1/1000 dilution.

Lane 1 : Histone H3 (ubiquitinated K23) peptide (aa20-29).

Lane 2 : Histone H3 non- ubiquitinated peptide (aa22-31).

Lane 3 : Histone H3 full length recombinant protein.

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 was used as secondary antibody.

Blocking and diluting buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.