Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (AB326331)

Immunohistochemical analysis of paraffin-embedded (A) : Mouse adipose without alkaline phosphatase treatment (B) : Mouse adipose with alkaline phosphatase treatment tissue labeling Hormone sensitive lipase/HSL with ab326331 at 1/2000 (0.263 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) mouse adipose without alkaline phosphatase treatment, no staining on (B) mouse adipose with alkaline phosphatase treatment.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (AB326331)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized :
Undifferentiated 3T3-L1 (mouse embryonic fibroblast)
3T3-L1 adipocytes treated with 10µM isoproterenol for 20 mins
3T3-L1 adipocytes treated with 10µM isoproterenol for 20 mins then treated with phosphatase at 37℃ for 1 hour
labelling Hormone sensitive lipase/HSL with ab326331 at 1/2000 (0.263 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Magenta).

Confocal image showing the increased cytoplasmic staining in 3T3-L1 adipocytes (shown in magenta) treated with 10µM isoproterenol for 20 mins, the signal was decreased afater treatment with Alkaline phosphatase at 37℃ for 1 hour. The counterstain was observed in green. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lipid Droplets Green Fluorescence Assay Kit with BODIPY493/503 was used to counterstain tubulin (Green). The Nuclear counterstain was DAPI (Blue).

• WB

Lab

Western blot - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (AB326331)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (ab326331) at 1/1000 dilution

Lane 1:

Undifferentiated 3T3-L1 (mouse embryonic fibroblast) whole cell lysate 20 µg (untreated membrane)

Lane 2:

3T3-L1 differentiated for 4 days, then treated with 10 µM Isoproterenol for 20 minutes whole cell lysate 20 µg (untreated membrane)

Lane 3:

3T3-L1 differentiated for 4 days, then treated with 10 µM Isoproterenol for 20 minutes whole cell lysate 20 µg (alkaline phosphatase treated membrane)

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: 83 kDa,36 kDa

false

Exposure time: 180s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (AB326331)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Hormone sensitive lipase/HSL with ab326331 at 1/2000 (0.263 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Low expression tissue : no staining on mouse lung.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Dot

Lab

Dot Blot - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (AB326331)

Dot blot analysis of Hormone sensitive lipase/HSL using ab326331 at 1 : 1000 (0.525 µg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane 1 : Hormone sensitive lipase/HSL (phospho S660) peptide

Lane 2 : Hormone sensitive lipase/HSL non-phospho peptide

Lane 3 : Hormone sensitive lipase/HSL (phospho S659) peptide

Lane 4 : Hormone sensitive lipase/HSL (phospho S659 + S660) peptide

Exposure time : 180 seconds.

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

All lanes:

Dot Blot - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (ab326331) at 1/1000 dilution

Lane 1:

Hormone sensitive lipase/HSL (phospho S660) peptide

Lane 2:

Hormone sensitive lipase/HSL non-phospho peptide

Lane 3:

Hormone sensitive lipase/HSL (phospho S659) peptide

Lane 4:

Hormone sensitive lipase/HSL (phospho S659 + S660) 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: 180s

• WB

Lab

Western blot - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (AB326331)

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (83KDa); Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Hormone sensitive lipase/HSL (phospho S660) antibody [EPR30648-593] (ab326331) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate 5 µg (untreated membrane)

Lane 2:

293T cells transfected with a mouse wild-type HSL expression vector containing a myc-His-tag® whole cell lysate 5 µg (untreated membrane)

Lane 3:

293T cells transfected with a mouse wild-type HSL expression vector containing a myc-His-tag®, treated with 100nM Calyculin A for 30 minutes whole cell lysate 5 µg (untreated membrane)

Lane 4:

293T cells transfected with a mouse HSL (mutation S660A) expression vector containing a myc-His-tag®, treated with 100nM Calyculin A for 30 minutes whole cell lysate 5 µg (alkaline phosphatase treated membrane)

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: 83 kDa,36 kDa

false

Exposure time: 8s