Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680)

3 Images

• WB

AbReview45305****

Western blot - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680) (AB175772)

Blocking : 1 hour at room temperature with 5% Milk in TBST.

Diluent : 2% Milk in TBST

13% SDS-PAGE gel used.

Secondary antibody - donkey anti-rabbit Alexa Fluor 680 (ab175772)

All lanes:

Western blot - Anti-p53 (phospho S6) antibody [Y179] (<a href='/en-us/products/primary-antibodies/p53-phospho-s6-antibody-y179-ab32132'>ab32132</a>) at 1/500 dilution

Lane 1:

DMSO treated HCT116 cell line at 20 µg

Lane 2:

10 µM irinotecan (CPT-11) treated HCT116 cell line for 24 hours at 20 µg

Secondary

All lanes:

Western blot - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680) (ab175772) at 1/1000 dilution

Predicted band size: 43 kDa

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Exposure time: 3min

This image is courtesy of an Abreview submitted by Christian Marx

• WB

Lab

Western blot - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680) (AB175772)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Milk before being incubated with ab18251 overnight at 4°C. Antibody binding was detected using ab175772 at a 1 : 10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (<a href='/en-us/products/primary-antibodies/alpha-tubulin-antibody-loading-control-ab18251'>ab18251</a>) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 5 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 2 µg

Lane 4:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 1 µg

Secondary

All lanes:

Western blot - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680) (ab175772) at 1/10000 dilution

Predicted band size: 36 kDa

Observed band size: 52 kDa

false

• ELISA

Lab

ELISA - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680) (AB175772)

Cross-reactivity of the polyclonal secondary antibody ab182020 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 μg/ml (50 μl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. ab182020 was then added starting at 1 μg/ml and gradually diluted 1/4 (50 μl/well), followed by incubation for 2h. For the detection Goat anti-Donkey IgG H&L (HRP) (ab6988) was used at 1/20,000 dilution (50 μl/well), followed by incubation for 1h at RT.

For the batch tested, ab182020 showed a cross-reactivity below 2% towards human IgG, mouse IgG, rat IgG, goat IgG and chicken IgY.

This data was developed using the unconjugated antibody (ab182020).