Anti-Collagen I antibody [EPR22894-89]

• WB

Lab

Western blot - Anti-Collagen I antibody [EPR22894-89] (AB260043)

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.

All lanes:

Western blot - 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:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/25000 dilution

Lane 3:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 139 kDa

false

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Collagen I antibody [EPR22894-89] (AB260043)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HFF-1 (Human skin fibroblast) cells labelling Collagen I with ab260043 at 1/500 (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 anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab260043).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Collagen I antibody [EPR22894-89] (AB260043)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling Collagen I with ab260043 at 1/500 (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 anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab260043).

• WB

Lab

Western blot - Anti-Collagen I antibody [EPR22894-89] (AB260043)

Blocking buffer and concentration : 5% NFDM/TBST.  Diluting buffer and concentration : 5% NFDM/TBST. Exposure Time : ab260043 : 20 seconds, ab255809 : 180 seconds. ab181602 was used as loading control. Compared with ab260043, ab255809 has lower background. We recommend ab255809 as an alternative for western blotting. For better use ab255809, we recommend loading higher amount of lysate or using lower antibody dilution.

All lanes:

Western blot - Anti-Collagen I antibody [EPR22894-89] (ab260043) at 1/1000 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate

Lane 3:

MDA-MB-231(Human breast adenocarcinoma epithelial cell) supernatant lysate

Lane 4:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate

Lane 5:

SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 139 kDa

Observed band size: 220 kDa,35 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-Collagen I antibody [EPR22894-89] (AB260043)

Collagen I was immunoprecipitated from 0.35 mg Mouse lung lysate 10ug with ab260043 at 1/30 dilution (2ug 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 10ug

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab260043).

All lanes:

Immunoprecipitation - Anti-Collagen I antibody [EPR22894-89] (ab260043)

Predicted band size: 139 kDa

false

• WB

Lab

Western blot - Anti-Collagen I antibody [EPR22894-89] (AB260043)

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:

Western blot - 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:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/25000 dilution

Predicted band size: 139 kDa

Observed band size: 220 kDa,35 kDa

false