Anti-STT3A antibody [EPR29178-10] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-STT3A antibody [EPR29178-10] - BSA and Azide free (AB320832)

This data was developed using ab320831, the same antibody clone in a different buffer formulation.

STT3A was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) whole cell lysate with ab320831 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab320831 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

In Immunoprecipitation, ab320831 was shown to bind specifically to STT3A. Target of interest was enriched at 60 kDa in wild-type HEK-293T cell lysates (lane 2) with no signal observed at this size in STT3A knockout cell line (lane 5) (lane 5, knockout cell line ab266320 / knockout cell lysate ab259164).
The bands above 60kDa are likely to be aggregation, and are absent in the KO samples. Unlike the Western Blot data, these samples have been boiled. ab181602 was used as a GAPDH loading control.

All lanes:

Immunoprecipitation - Anti-STT3A antibody [EPR29178-10] (<a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a>) at 1/1000 dilution

Lane 1:

Parental HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

Parental HEK-293T (human embryonic kidney epithelial cell) 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 <a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in Parental HEK-293T whole cell lysate

Lane 4:

STT3A KO HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 5:

Immunoprecipitation - Human STT3A knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-stt3a-knockout-hek-293t-cell-line-ab266320'>ab266320</a>)

Lane 6:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in STT3A KO HEK-293T 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: 60 kDa

false

Exposure time: 84s

• IP

Lab

Immunoprecipitation - Anti-STT3A antibody [EPR29178-10] - BSA and Azide free (AB320832)

This data was developed using ab320831, the same antibody clone in a different buffer formulation.

STT3A was immunoprecipitated from 0.35 mg MEF (mouse embryo fibroblast) whole cell lysate with ab320831 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab320831 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

The bands above 60kDa are likely to be aggregation. Unlike the Western Blot data, these samples have been boiled.

All lanes:

Immunoprecipitation - Anti-STT3A antibody [EPR29178-10] (<a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a>) at 1/1000 dilution

Lane 1:

MEF (mouse embryo fibroblast) whole cell lysate at 10 µg

Lane 2:

MEF (mouse embryo fibroblast) 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 <a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in MEF 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: 60 kDa

false

Exposure time: 84s

• WB

Supplier Data

Western blot - Anti-STT3A antibody [EPR29178-10] - BSA and Azide free (AB320832)

This data was developed using ab320831, the same antibody clone in a different buffer formulation.

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

In Western blot, ab320831 was shown to bind specifically to STT3A. Target of interest was observed at 60 kDa in wild-type HEK-293T cell lysates (lane 1) with no signal observed at this size in STT3A knockout cell line (lane 2) (lane 2, knockout cell line ab266320 / knockout cell lysate ab259164).

The molecular weight observed is consistent with what has been described in the literature (PMID : 22157935).

Samples are non-boiled as boiling may cause protein aggregation.

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

All lanes:

Western blot - Anti-STT3A antibody [EPR29178-10] (<a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

STT3A knockout HEK-293T whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HeLa (human cervical adenocarcinoma epithelial cell) 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: 60 kDa,36 kDa

false

Exposure time: 59s

• WB

Supplier Data

Western blot - Anti-STT3A antibody [EPR29178-10] - BSA and Azide free (AB320832)

This data was developed using ab320831, the same antibody clone in a different buffer formulation.

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

Samples are non-boiled as boiling may cause protein aggregation.

Exposure time : Lane 1 : 180 seconds; Lane 2 : 59 seconds.

All lanes:

Western blot - Anti-STT3A antibody [EPR29178-10] (<a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a>) at 1/1000 dilution

Lane 1:

Human lung tissue lysate at 20 µg

Lane 2:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 60 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-STT3A antibody [EPR29178-10] - BSA and Azide free (AB320832)

This data was developed using ab320831, the same antibody clone in a different buffer formulation.

Dot blot analysis of STT3A using ab320831 at 1 : 1000 (0.493 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : Human STT3A peptide
Lane2 : Human STT3B peptide

Exposure time : 180 seconds.

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

This antibody does not cross-react with human STT3B peptide by dot blot.

Lane 1:

Human STT3A peptide

Lane 2:

Human STT3B peptide

false

Exposure time: 180s