Anti-TSG6 antibody [EPR17582-45] (ab267469) at 1/1000 dilution + Rat amniotic membrane tissue lysate at 20 µg

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

Predicted band size: 31 kDa
Observed band size: 120,36 kDa why is the actual band size different from the predicted?

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

Band of 36KDa represents the monomeric TSG6. Band of 120KDa likely represents a stable covalent complex formed between TSG6 and one of the heavy chains of inter-α-inhibitor or pre-α-inhibitor (PMID: 22351758, 11420253).

The expression profile observed is consistent with what has been described in the literature (PMID: 22351758, 11420253, 19389798).

Exposure time: 3 minutes

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte) (treated with 100ng/ml lipopolysaccharide (LPS) for 1h, then add BFA for 4h) (Red) / Untreated control (Green) cells labelling TSG6 with ab267469 at 1/500 dilution (0.1ug) (Red, Green) 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 anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

TSG6 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) (treated with 100ng/mg LPS for 3 hours, then treated with BFA for 2 hours) whole cell lysate with ab267469 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267469 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (treated with 100ng/mg LPS for 3 hours, then treated with BFA for 2 hours) whole cell lysate 10 ug.

Lane 2: ab267469 IP in THP-1 (treated as above).

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267469 in THP-1 (treated as above) whole cell lysate

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

Exposure time: 41 seconds

This blot was developed using a higher sensitivity ECL substrate.

Lane 1 : Anti-TSG6 antibody [EPR17582-45] (ab267469) at 1/1000 dilution
Lanes 2-3 : Anti-p75 NGF Receptor antibody [NORI138B] (ab271289) at 1/1000 dilution

Lane 1 : THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lane 2 : THP-1 (treated with 100ng/mg LPS for 3 hours, then treated with BFA for 2 hours), whole cell lysate
Lane 3 : A549 (human lung carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 31 kDa
Observed band size: 120,36 kDa why is the actual band size different from the predicted?

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

Band of 36KDa represents the monomeric TSG6. Band of 120KDa likely represents a stable covalent complex formed between TSG6 and one of the heavy chains of inter-α-inhibitor or pre-α-inhibitor (PMID: 22351758, 11420253).

The expression profile observed is consistent with what has been described in the literature (PMID: 22351758, 11420253, 19389798).

Negative control: A549 (PMID: 19389798).

Exposure time: 3 minutes