Anti-ERK5 (phospho T733) antibody [EPR28258-101]

• WB

Supplier Data

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (AB323508)

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

In Western blot, ab323508 was shown to bind specifically to ERK5 (phospho T733). Target of interest was observed at 115 kDa in treated wild-type HeLa cell lysates (lanes -2-4) with no signal observed at this size in treated ERK5 knockout cell line (lanes 6-8) (lanes 5-8, knockout cell line ab265508 / knockout cell lysate ab258042).

Lane 1-8 on the left : untreated membrane;
Lane 1-8 on the right : membrane treated with alkaline phosphatase.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-ERK5 antibody [EP791Y] - Total protein control (ab40809) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (ab323508) at 1/1000 dilution

Lane 1:

Untreated parental HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

Lane 2:

Parental HeLa treated with 0.4M Sorbitol for 30min whole cell lysate at 20 µg with NFDM/TBST

Lane 3:

Parental HeLa treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg with NFDM/TBST

Lane 4:

Parental HeLa treated with 0.4M Sorbitol and 100nM Calyculin A for 30min whole cell lysate at 20 µg with NFDM/TBST

Lane 5:

Untreated ERK5 knockout HeLa whole cell lysate at 20 µg with NFDM/TBST

Lane 6:

ERK5 knockout HeLa treated with 0.4M Sorbitol for 30min whole cell lysate at 20 µg with NFDM/TBST

Lane 7:

ERK5 knockout HeLa treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg with NFDM/TBST

Lane 8:

ERK5 knockout HeLa treated with 0.4M Sorbitol and 100nM Calyculin A for 30min whole cell lysate at 20 µg with NFDM/TBST

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: 115 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (AB323508)

Lane 1-4 on the left : untreated membrane;
Lane 1-4 on the right : membrane treated with alkaline phosphatase.

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

In Western blot, Anti-ERK5 antibody [EP791Y] - Total protein control (ab40809) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (ab323508) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg with NFDM/TBST

Lane 2:

NIH/3T3 treated with 0.4M Sorbitol for 30min whole cell lysate at 20 µg with NFDM/TBST

Lane 3:

NIH/3T3 treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg with NFDM/TBST

Lane 4:

NIH/3T3 treated with 0.4M Sorbitol and 100nM Calyculin A for 30min whole cell lysate at 20 µg with NFDM/TBST

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: 115 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (AB323508)

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In Western blot, Anti-ERK5 antibody [EP791Y] - Total protein control (ab40809) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (ab323508) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells co-transfected with a human ERK5 WT expression vector containing a myc-His-tag® and a rat MEK5(S311D/T315D mutation)) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg with NFDM/TBST

Lane 2:

293T (human embryonic kidney epithelial cell) cells co-transfected with a human ERK5 (T733A mutation) expression vector containing a myc-His-tag® and a rat MEK5(S311D/T315D mutation)) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg with NFDM/TBST

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: 115 kDa

false

Exposure time: 70s

• Dot

Supplier Data

Dot Blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (AB323508)

Dot blot analysis of ERK5 (phospho T733) using ab323508 at 1 : 1000 (0.51 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : ERK5 (phospho T733) peptide
Lane2 : ERK5 non-phospho peptide

Exposure time : 15 seconds.

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

All lanes:

Dot Blot - Anti-ERK5 (phospho T733) antibody [EPR28258-101] (ab323508) at 1/1000 dilution

Lane 1:

ERK5 (phospho T733) peptide

Lane 2:

ERK5 non-phospho peptide

Secondary

All lanes:

Dot 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

false

Exposure time: 15s