重组Anti-SIRT5抗体[EPR23787-116]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Immunohistochemical analysis of paraffin-embedded Human ovarian adenocarcinoma tissue labeling SIRT5 with ab259967 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human ovarian adenocarcinoma (PMID 31456942). 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (Human hepatocellular carcinoma epithelial cell) cells labelling SIRT5 with ab259967 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling SIRT5 with ab259967 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human liver (PMID 29491006). 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

Unknown

Immunoprecipitation - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

SIRT5 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab259967 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259967 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 ug

Lane 2 : ab259967 IP in HepG2 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab259967 in HepG2 whole cell lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-SIRT5 antibody [EPR23787-116] (ab259967)

Predicted band size: 33 kDa

Observed band size: 30 kDa

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• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Hepa1-6 (Mouse hepatoma epithelial cell) cells labelling SIRT5 with ab259967 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling SIRT5 with ab259967 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse liver (PMID 29491006). 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling SIRT5 with ab259967 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat liver (PMID 29491006). 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

Unknown

Immunoprecipitation - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

SIRT5 was immunoprecipitated from 0.35 mg Mouse liver tissue lysate with ab259967 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259967 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse liver tissue lysate 10 ug

Lane 2 : ab259967 IP in Mouse liver tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab259967 in mouse liver tissue lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-SIRT5 antibody [EPR23787-116] (ab259967)

Predicted band size: 33 kDa

Observed band size: 30 kDa

false

• WB

Lab

Western blot - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Western blot : Anti-SIRT5 antibody [EPR23787-116] (ab259967) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab259967 was shown to bind specifically to SIRT5. A band was observed at 33 kDa in wild-type A549 cell lysates with no signal observed at this size in SIRT5 knockout cell line. To generate this image, wild-type and SIRT5 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-SIRT5 antibody [EPR23787-116] (ab259967) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human SIRT5 knockout A549 cell line (<a href='/products/cell-lines/human-sirt5-knockout-a549-cell-line-ab282634'>ab282634</a>)

Lane 2:

SIRT5 knockout A549 cell lysate at 20 µg

Lane 3:

PC-3 cell lysate at 20 µg

Lane 4:

MCF7 Membrane Prep cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 33 kDa

false

• WB

Unknown

Western blot - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-SIRT5 antibody [EPR23787-116] (ab259967) at 1/1000 dilution

Lane 1:

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

Lane 2:

Huh7 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 5:

Hepa1-6 (mouse hepatoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 7:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 30 kDa

false

• WB

Unknown

Western blot - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-SIRT5 antibody [EPR23787-116] (ab259967) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 10 µg

Lane 2:

Human heart tissue lysate at 10 µg

Secondary

All lanes:

VeriBlot for IP secondary antibody(HRP)(<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 33 kDa

Observed band size: 30 kDa

false

• WB

Unknown

Western blot - Anti-SIRT5 antibody [EPR23787-116] (AB259967)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-SIRT5 antibody [EPR23787-116] (ab259967) at 1/1000 dilution

Lane 1:

Mouse liver tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Rat liver tissue lysate at 20 µg

Lane 4:

Rat heart tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 30 kDa

false