Anti-Id1 antibody [EPR24304-18A]

• WB

Lab

Western blot - Anti-Id1 antibody [EPR24304-18A] (AB283650)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 11034201, PMID : 1644289).

Exposure time : 3 minutes

All lanes:

Western blot - Anti-Id1 antibody [EPR24304-18A] (ab283650) at 1/1000 dilution

Lane 1:

MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 3:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 16 kDa

Observed band size: 14-16 kDa

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• WB

Lab

Western blot - Anti-Id1 antibody [EPR24304-18A] (AB283650)

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

This blot was developed using a higher sensitivity ECL substrate.

In materials from rats, ab283650 is suitable only for WB application.

Exposure time : 92 seconds

All lanes:

Western blot - Anti-Id1 antibody [EPR24304-18A] (ab283650) at 1/1000 dilution

Lane 1:

Untreated PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 2:

PC-12 treated with 50 ng/ml EGF (<a href='/en-us/products/proteins-peptides/recombinant-human-egf-protein-animal-free-ab9697'>ab9697</a>) for 48 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 16 kDa

Observed band size: 14-16 kDa

false

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-Id1 antibody [EPR24304-18A] (AB283650)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HCT 116 (Human colorectal carcinoma epithelial cell) cells labelling Id1 with ab283650 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG (DyLight^® 488, ab98507) at 1/500 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-Id1 antibody [EPR24304-18A] (AB283650)

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

Exposure time : 8 seconds

All lanes:

Western blot - Anti-Id1 antibody [EPR24304-18A] (ab283650) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 2:

HEK-293T transfected with human Id1 (WT) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 16 kDa

Observed band size: 14-16 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Id1 antibody [EPR24304-18A] (AB283650)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HCT 116 cells labelling Id1 with ab283650 at 1/50 (9.64 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear and weakly cytoplasmic staining in HCT 116 cell line. is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• WB

Lab

Western blot - Anti-Id1 antibody [EPR24304-18A] (AB283650)

False colour image of Western blot : Anti-Id1 antibody [EPR24304-18A] 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, ab283650 was shown to bind specifically to Id1. A band was observed at 16 kDa in wild-type A549 cell lysates with no signal observed at this size in ID1 knockout cell line ab269603 (knockout cell lysate ab272442). To generate this image, wild-type and ID1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Id1 antibody [EPR24304-18A] (ab283650) at 1/1000 dilution

Lane 1:

Wild-type A549 vehicle control nicotine (0 uM, 18 h) cell lysate at 20 µg

Lane 2:

Wild-type A549 treated nicotine (1 uM, 18 h) cell lysate at 20 µg

Lane 3:

ID1 knockout A549 vehicle control nicotine (0 uM, 18 h) cell lysate at 20 µg

Lane 4:

ID1 knockout A549 treated nicotine (1 uM, 18 h) cell lysate at 20 µg

Lane 5:

PC-3 cell lysate at 20 µg

Lane 6:

HeLa cell lysate at 20 µg

Lane 7:

HeLa nuclear cell lysate at 20 µg

Observed band size: 16 kDa

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