Anti-ADAM10 antibody [EPR5622]

• IP

Lab

Immunoprecipitation - Anti-ADAM10 antibody [EPR5622] (AB124695)

ab124695 (purified) at 1 : 30 dilution (2ug) immunoprecipitating ADAM-10 in LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate 10ug.
Lane 1 (input) : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab124695 & LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate 10ug
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab170952 in HeLa LNCaP (Human prostate carcinoma epithelial cell) whole cell lysateFor western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-ADAM10 antibody [EPR5622] (ab124695)

Predicted band size: 84 kDa

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• WB

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Western blot - Anti-ADAM10 antibody [EPR5622] (AB124695)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-ADAM10 antibody [EPR5622] (ab124695) at 0.09 µg/mL

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

LNCaP (Human prostate carcinoma 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

Predicted band size: 21 kDa,84 kDa

Observed band size: 19 kDa

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• WB

Lab

Western blot - Anti-ADAM10 antibody [EPR5622] (AB124695)

False colour image of Western blot : Anti-ADAM10 antibody [EPR5622] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab124695 was shown to bind specifically to ADAM10. A band was observed at 95/120 kDa in wild-type Jurkat cell lysates with no signal observed at this size in ADAM10 knockout cell line. The band at 120 kDa is likely to be the precursor and the band at 90 kDa is likely to be the active form of ADAM10. To generate this image, wild-type and ADAM10 knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-ADAM10 antibody [EPR5622] (ab124695) at 1/1000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 10 µg

Lane 2:

ADAM10 knockout Jurkat cell lysate at 10 µg

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

U-2 OS cell lysate at 20 µg

Observed band size: 95-120 kDa

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• WB

Lab

Western blot - Anti-ADAM10 antibody [EPR5622] (AB124695)

Lane 1 : Wild type HAP1 whole cell lysate (40 μg)
Lane 2 : Empty lane
Lane 3 : ADAM10 knockout HAP1 whole cell lysate (40 μg)
Lane 4 : U20S whole cell lysate (40 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab124695 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124695 was shown to recognize ADAM10 when ADAM10 knockout samples were used, along with additional cross-reactive bands. Wild-type and ADAM10 knockout samples were subjected to SDS-PAGE. ab124695 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ADAM10 antibody [EPR5622] (ab124695)

Predicted band size: 84 kDa

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• WB

Lab

Western blot - Anti-ADAM10 antibody [EPR5622] (AB124695)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of Jurkat whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab124695 (1/10,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab124695 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-ADAM10 antibody [EPR5622] (ab124695) at 1/10000 dilution

All lanes:

Jurkat whole cell lysate at 15 µg with NDFM/TBST

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

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Exposure time: 40s

• WB

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Western blot - Anti-ADAM10 antibody [EPR5622] (AB124695)

84 kDa band is pro-ADAM10, smaller band at around 65 kDa is mature ADAM10 protein.

All lanes:

Western blot - Anti-ADAM10 antibody [EPR5622] (ab124695) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

RAW 264.7 cell lysate at 10 µg

Lane 3:

LnCaP cell lysate at 10 µg

Lane 4:

MCF-7 cell lysate at 10 µg

Predicted band size: 84 kDa

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• WB

Lab

Western blot - Anti-ADAM10 antibody [EPR5622] (AB124695)

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

The antibody detects a nonspecific band close to target band when detecting tissue samples. It is recommended to optimize experimental conditions by increasing the sample loading amount, using a lower antibody dilution ratio, and employing femtogram-level sensitivity substrates.

All lanes:

Western blot - Anti-ADAM10 antibody [EPR5622] (ab124695) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 20 µg

Lane 3:

Mouse heart tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Rat lung tissue lysate at 20 µg

Lane 6:

Rat heart tissue 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: 70 kDa,80 kDa

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Exposure time: 100s

• WB

Unknown

Western blot - Anti-ADAM10 antibody [EPR5622] (AB124695)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-ADAM10 antibody [EPR5622] (ab124695) at 0.05 µg/mL

Lane 1:

RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

Rat brain 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

Predicted band size: 84 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-ADAM10 antibody [EPR5622] (AB124695)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.