Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HFF-1 (Human skin fibroblast) cells labeling Collagen I with ab260043 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HFF-1 cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labeling Collagen I with ab260043 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

All lanes : Anti-Collagen I antibody [EPR22894-89] (ab260043) at 1/5000 dilution

Lane 1 : HFF-1 (human skin fibroblast) whole cell lysate at 20 µg
Lane 2 : HFF-1 culture supernatant at 10 µl

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/25000 dilution

Predicted band size: 139 kDa
Observed band size: 220,35 kDa why is the actual band size different from the predicted?

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

Procollagen I (220 kDa) and cleaved C-propeptide (35 kDa) are observed.

The molecular weight observed is consistent with what has been described in the literature (PMID: 23940311). 

Exposure time: Lane 1: 1 second; Lane 2: 3 seconds.

All lanes : Anti-Collagen I antibody [EPR22894-89] (ab260043) at 1/1000 dilution

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) culture supernatant at 10 µl
Lane 2 : Rat lung organ culture supernatant at 10 µl
Lane 3 : Human lung tissue lysate at 10 µg

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/25000 dilution
Lane 3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 139 kDa

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

Cleaved collagen I C-propeptide (35 kDa) is observed.

The molecular weight observed is consistent with what has been described in the literature (PMID: 23940311). 

Exposure time: Lanes 1-2: 3 seconds; Lane 3: 136 seconds.

Collagen I was immunoprecipitated from 0.35 mg mouse lung lysate 10µg with ab260043 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab260043 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: Mouse lung lysate 10µg

Lane 2: ab260043 IP in mouse lung lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab260043 in mouse lung lysate

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

Exposure time: 1 second.

 

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labeling Collagen I with ab260043 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HFF-1 (Human skin fibroblast) cells labeling Collagen I with ab260043 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.