Anti-KMT3A / HYPB / HIF-1 antibody [EPR28172-44]

• IP

Supplier Data

Immunoprecipitation - Anti-KMT3A / HYPB / HIF-1 antibody [EPR28172-44] (AB324102)

KMT3A / HYPB / HIF-1 was immunoprecipitated from 0.35 mg ab324102 at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab324102 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324102 at 1/1000 dilution.

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-KMT3A / HYPB / HIF-1 antibody [EPR28172-44] (ab324102) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 2:

ab324102 at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324102 in Hela whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 120-350 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-KMT3A / HYPB / HIF-1 antibody [EPR28172-44] (AB324102)

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

This antibody was tested on fresh and frozen Hela and 293T whole cell lysate and it was concluded that SETD2 (KMT3A / HYPB / HIF-1) is the easy target for degradation.

To minimize protein degradation, cells (lanes 3-4) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

Exposure time : Lanes 1-2 : 180 seconds; Lanes 3-4 : 81 seconds.

All lanes:

Western blot - Anti-KMT3A / HYPB / HIF-1 antibody [EPR28172-44] (ab324102) at 1/1000 dilution

Lanes 1 and 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 4:

293T (human embryonic kidney 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

Observed band size: 120-350 kDa

false

• WB

Supplier Data

Western blot - Anti-KMT3A / HYPB / HIF-1 antibody [EPR28172-44] (AB324102)

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

In Western blot, ab324102 was shown to bind specifically to SETD2 (KMT3A / HYPB / HIF-1). Target of interest was observed at 120-350 kDa in wild-type Hela cell lysates (lane 1) with no signal observed at this size in SETD2 (KMT3A / HYPB / HIF-1) knockout cell line (lane 2) (lane 2, knockout cell line ab257273).

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-KMT3A / HYPB / HIF-1 antibody [EPR28172-44] (ab324102) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SETD2 (KMT3A / HYPB / HIF-1) knockout HeLa cell lysate knockout HeLa 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: 120-350 kDa,124 kDa

true

Exposure time: 180s