All lanes : Anti-PUS1 antibody [EPR20181] (ab203010) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : PUS1 knockout HEK-293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab203010 observed at 45 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab203010 was shown to react with PUS1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266091 (knockout cell lysate ab258158) was used. Wild-type HEK-293T and PUS1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab203010 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling PUS1 with ab203010 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line.

The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

This antibody was not successful when we used it on RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells in ICC application. This antibody was not tested on rat cells in ICC.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling PUS1 with ab203010 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mitochondrial staining on HeLa cell line.

The nuclear counterstain is DAPI (blue). COXIV is detected with ab33985 (Anti-COX IV(mouse mAb)) at 1/1000 dilution followed by Goat anti-mouse IgG (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab203010 at 1/500 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab33985 (anti-COX IV(mouse mAb)) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Anti-PUS1 antibody [EPR20181] (ab203010) at 1/1000 dilution + Human skeletal muscle lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 47 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PUS1 antibody [EPR20181] (ab203010) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) mitochondria lysate
Lane 2 : HeLa cytoplasm fraction lysate
Lane 3 : A431 (Human epidermoid carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47 kDa
Observed band size: 47 kDa


Exposure time: 8 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in UniProt.

Anti-PUS1 antibody [EPR20181] (ab203010) at 1/1000 dilution + HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47 kDa
Observed band size: 44,47 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on sequence alignment, the antibody can recognize 2 isoforms, the predicted MW are 47kDa and 44kDa, respectively [PMID: 17056637].

All lanes : Anti-PUS1 antibody [EPR20181] (ab203010) at 1/2000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat spleen lysate
Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 5 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 7 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47 kDa
Observed band size: 47 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 10 seconds; Lane 3: 3minutes; Lane 4,5,6 and 7: 10 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling PUS1 with ab203010 at 1/400 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

PUS1 was immunoprecipitated from 0.35 mg of HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate with ab203010 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab203010 at 1/1000 dilution. VeriBlot for IP Detection Reaction (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HEK-293 whole cell lysate, 10µg (Input).

Lane 2: ab203010 IP in HEK-293 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab203010 in HEK-293 whole cell lysate.

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

Exposure time: 5 seconds.