Anti-SHP2 antibody [EPR26539-45]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HEK293 tissue labeling SHP2 with ab300579 at 1/200 (1.22 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in wild-type HEK293T cells and no staining in PTPN11 (SHP2) knockout HEK293T cells. The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling SHP2 with ab300579 at 1/50 (9.76 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in human tonsil (PMID : 18728972). The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PTPN11 KO HEK293T (PTPN11 KO knock out human embryonic kidney epithelial cell) (ab266450) cells labelling SHP2 with ab300579 at 1/50 (9.76 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in wildtype HEK293T cell line, while showing no staining in PTPN11 knockout HEK293T cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-SHP2 antibody [EPR26539-45] (AB300579)

SHP2 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell), whole cell lysate 10 ug with ab300579 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300579 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell), whole cell lysate 10 ug

Lane 2 : ab300579 IP in Hela whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab300579 in Hela whole cell lysate

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

Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-SHP2 antibody [EPR26539-45] (ab300579) at 1/1000 dilution

All lanes:

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

false

Exposure time: 180s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling SHP2 with ab300579 at 1/50 (9.76 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in mouse testis (PMID : 24123360). The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling SHP2 with ab300579 at 1/50 (9.76 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in rat testis (PMID : 24123360). The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling SHP2 with ab300579 at 1/50 (9.76 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-SHP2 antibody [EPR26539-45] (AB300579)

SHP2 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10ug with ab300579 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300579 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10ug.

Lane 2 : ab300579 IP in NIH/3T3 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab300579 in NIH/3T3 whole cell lysate.

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

Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-SHP2 antibody [EPR26539-45] (ab300579) at 1/1000 dilution

All lanes:

NIH/3T3 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

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-SHP2 antibody [EPR26539-45] (AB300579)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

The bands beneath the target band (72 kDa) are likely to be degraded target fragments.

All lanes:

Western blot - Anti-SHP2 antibody [EPR26539-45] (ab300579) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Human heart tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG (Merck DC03L) at 1/2000 dilution

Observed band size: 72 kDa

true

Exposure time: 26s

• WB

Lab

Western blot - Anti-SHP2 antibody [EPR26539-45] (AB300579)

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

Exposure time Lane 1-8 : 180s

Exposure time Lane 9-10 : 1s

Exposure time Lane 11-16 : 15s

ab300579 is less sensitive than ab187040 in WB. We suggest customers choosing ab187040 for WB test.

Lanes 1 - 8:

Western blot - Anti-SHP2 antibody [EPR26539-45] (ab300579) at 1/1000 dilution

Lanes 9 - 16:

Western blot - Anti-SHP2 antibody [EPR17829-9] (<a href='/en-us/products/primary-antibodies/shp2-antibody-epr17829-9-ab187040'>ab187040</a>) at 1/1000 dilution

Lanes 1 and 9:

Human cerebellum tissue lysate, 20 μg

Lanes 2 and 10:

Human heart tissue lysate, 20 μg

Lanes 3 and 11:

Mouse ovary tissue lysate, 20 μg

Lanes 4 and 12:

Mouse embryo tissue lysate, 20 μg

Lanes 5 and 13:

Mouse brain tissue lysate, 20 μg

Lanes 6 and 14:

Mouse heart tissue lysate, 20 μg

Lanes 7 and 14:

Rat brain tissue lysate, 20 μg

Lanes 8 and 16:

Rat heart tissue lysate, 20 μg

Secondary

Lanes 1, 2, 9 and 10:

Goat Anti-Rabbit IgG (HRP), minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15 and 16:

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: 68 kDa

Observed band size: 72 kDa

false

• WB

Supplier Data

Western blot - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysates at 20 µg per lane.In Western blot, ab300579 was shown to bind specifically to PTPN11 (SHP2). A band was observed at 72 kDa in wild-type HEK293T cell lysates with no signal observed at this size in PTPN11 (SHP2) knockout cell line ab266450 (knockout cell lysate ab257618).

All lanes:

Western blot - Anti-SHP2 antibody [EPR26539-45] (ab300579) at 1/1000 dilution

Lane 1:

Wild-type HEK293T (human embryonic kidney epithelial cell), whole cell lysate 20 µg

Lane 2:

PTPN11 (SHP2) knockout HEK293T whole cell lysate 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate 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 1/100000 dilution

Observed band size: 72 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-SHP2 antibody [EPR26539-45] (AB300579)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

The bands beneath the target band (72 kDa) are likely to be degraded target fragments.

All lanes:

Western blot - Anti-SHP2 antibody [EPR26539-45] (ab300579) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Rat brain tissue lysate at 20 µg

Lane 4:

Rat heart tissue 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 1/100000 dilution

Observed band size: 72 kDa

true

Exposure time: 81s

• WB

Supplier Data

Western blot - Anti-SHP2 antibody [EPR26539-45] (AB300579)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-SHP2 antibody [EPR26539-45] (ab300579) at 1/1000 dilution

All lanes:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 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 1/100000 dilution

Observed band size: 72 kDa

false

Exposure time: 48s