Anti-HINT1 antibody [EPR5108]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HINT1 antibody [EPR5108] (AB124912)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial cell) cells labeling HINT1 with purified ab124912 at 1/1000 dilution (2 μg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HINT1 antibody [EPR5108] (AB124912)

ab124912, at 1/250 dilution, staining HINT1 in paraffin embedded colonic adenocarcinoma tissue by immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HINT1 antibody [EPR5108] (AB124912)

ab124912, at 1/250 dilution, staining HINT1 in paraffin embedded breast carcinoma tissue by immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-HINT1 antibody [EPR5108] (AB124912)

Lanes 1-3 : Merged signal (red and green). Green - ab124912 observed at 17 kDa. Red - loading control ab8245 observed at 36 kDa.

ab124912 Anti-HINT1 antibody [EPR5108] was shown to specifically react with HINT1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265776 (knockout cell lysate ab257465) was used. Wild-type and HINT1 knockout samples were subjected to SDS-PAGE. ab124912 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HINT1 antibody [EPR5108] (ab124912) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human HINT1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-hint1-knockout-hela-cell-line-ab265776'>ab265776</a>)

Lane 2:

HINT1 knockout HeLa cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 14 kDa

Observed band size: 17 kDa

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• WB

Lab

Western blot - Anti-HINT1 antibody [EPR5108] (AB124912)

Western blot : Rabbit Monoclonal[EPR5108] to HINT1 ab124912 staining at 1/500 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 14 kDa in Wild-type HeLa cell lysates with no signal observed at this size in HINT1 knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-HINT1 antibody [EPR5108] (ab124912) at 1/500 dilution

Lane 1:

Wild-type HeLa at 20 µg

Lane 2:

Western blot - Human HINT1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-hint1-knockout-hela-cell-line-ab265776'>ab265776</a>) at 20 µg

Lane 3:

Jurkat at 20 µg

Lane 4:

HeLa Membrane at 20 µg

Lane 5:

EMPTY

Lane 6:

Western blot - Recombinant Human HINT1 protein (<a href='/en-us/products/proteins-peptides/recombinant-human-hint1-protein-ab87362'>ab87362</a>) at 0.1 µg

Secondary

Lanes 1 - 6:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 6:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa,37 kDa

false

• WB

Unknown

Western blot - Anti-HINT1 antibody [EPR5108] (AB124912)

All lanes:

Western blot - Anti-HINT1 antibody [EPR5108] (ab124912) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

MCF7 cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Lane 4:

Jurkat cell lysate at 10 µg

Predicted band size: 14 kDa

false