All lanes : Anti-FGF2 antibody [EPR20145-219] (ab208687) at 1/1000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 2 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 3 : SK-OV-3 (Human ovarian cancer 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: 30 kDa
Observed band size: 18,22,24 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2: 15 seconds; Lane 3: 5 seconds.

The MW observed is consistent with the literatures (PMID:18850066, PMID: 24827991).

 

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling FGF2 with ab208687 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and weak cytoplasmic staining on U-87 MG 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.

Anti-FGF2 antibody [EPR20145-219] (ab208687) at 1/10000 dilution + Recombinant human FGF2 active protein (aa143-288) at 0.01 µg

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

Predicted band size: 30 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-FGF2 antibody [EPR20145-219] (ab208687) at 1/1000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human prostate lysate
Lane 3 : Human fetal heart lysate
Lane 4 : Human testis lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 30 kDa
Observed band size: 18,22,24 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The MW observed is consistent with the literatures (PMID:18850066, PMID: 24827991).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-OV-3 (Human ovarian cancer cell line) cells labeling FGF2 with ab208687 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear weak cytoplasmic staining on SK-OV-3 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.

Flow cytometric analysis of 4% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling FGF2 with ab208687 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled 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.

FGF2 was immunoprecipitated from 0.35 mg of K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate with ab208687 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab208687 at 1/500 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: K562 whole cell lysate 10µg (Input).

Lane 2: ab208687 IP in K562 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208687 in K562 whole cell lysate.

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

Exposure time: 1 second.

ELISA analysis of Mouse FGF2 recombinant protein at 1000 ng/ml with ab208687. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.