Anti-SH3MD2 antibody [EPR30521-594]

• IP

Lab

Immunoprecipitation - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

SH3MD2 was immunoprecipitated from 0.35 mg HCT 116 (human colorectal carcinoma epithelial cell) fresh whole cell lysate with ab326568 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab326568 at 1/326568 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell) fresh whole cell lysate
Lane 2 : ab326568 IP in HCT 116 (human colorectal carcinoma epithelial cell) fresh whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab326568 in HCT 116 fresh whole cell lysate

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

Exposure time : 145 seconds.

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

All lanes:

Immunoprecipitation - Anti-SH3MD2 antibody [EPR30521-594] (ab326568) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 2:

ab326568 at 1/30 IP in HCT 116 (human colorectal carcinoma epithelial cell) fresh 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 ab326568 in HCT 116 fresh 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: 100 kDa

false

Exposure time: 145s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cells labelling SH3MD2 with ab326568 at 1/200 (2.515 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in mouse primary neural/glia cell (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain at 1/500 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1 : !000 dilution (Magenta).

-ve control 1 : ab326568 at 1/200 dilution, followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab11267 at 1/500 dilution, followed by ab150081 at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse splenocytes labelling SH3MD2 with ab326568 at 1/200 (2.515 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Low expression : Confocal image showing weak staining in mouse splenocytes (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab326568 at 1/200 dilution, followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• I-ELISA

Lab

Indirect ELISA - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

Indirect ELISA analysis of ab326568 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Untagged mouse SH3RF1 fragment;Untagged mouse SH3RF3 fragment.

Antigen concentration : 1000 ng/ml

This antibody does not cross-react with mouse SH3RF3.

• IP

Lab

Immunoprecipitation - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

SH3MD2 was immunoprecipitated from 0.35 mg Neuro-2a (mouse neuroblastoma neuroblast) fresh whole cell lysate with ab326568 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab326568 at 1/326568 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Neuro-2a (mouse neuroblastoma neuroblast) fresh whole cell lysate
Lane 2 : ab326568 IP in Neuro-2a (mouse neuroblastoma neuroblast) fresh whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab326568 in Neuro-2a fresh whole cell lysate

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

Exposure time : 145 seconds.

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

All lanes:

Immunoprecipitation - Anti-SH3MD2 antibody [EPR30521-594] (ab326568) at 1/1000 dilution

Lane 1:

Neuro-2a (mouse neuroblastoma neuroblast) fresh whole cell lysate at 10 µg

Lane 2:

ab326568 at 1/30 IP in Neuro-2a (mouse neuroblastoma neuroblast) fresh 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 ab326568 in Neuro-2a fresh 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: 100 kDa

false

Exposure time: 145s

• WB

Lab

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

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

Low expression : Daudi.

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-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (ab326568) at 1/1000 dilution

Lane 1:

NIH:OVCAR-3 (human ovary adenocarcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 2:

Daudi (human Burkitt's lymphoma lymphoblast) fresh 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: 100 kDa,36 kDa

false

Exposure time: 147s

• WB

Lab

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

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

Low expression : K-562.

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-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (ab326568) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 2:

K-562 (human chronic myelogenous leukemia lymphoblast) fresh 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: 100 kDa,36 kDa

false

Exposure time: 147s

• WB

Lab

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

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

Low expression : A20.

In lanes 2-3, the lysate was stored at -80°C prior to Western Blotting. The bands beneath the target band (100 kDa) are likely to be degradation products. In lane 1, To minimize protein degradation, cell was lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

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

false

Exposure time: 147s

• WB

Lab

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (AB326568)

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

Low expression : spleen (PMID : 9482736).

To minimize protein degradation, tissues 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-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (ab326568) at 1/1000 dilution

Lane 1:

Mouse brain fresh tissue lysate at 20 µg

Lane 2:

Mouse spleen fresh 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 1/20000 dilution

Observed band size: 100 kDa,36 kDa

false

Exposure time: 147s