All lanes : Anti-MTHFR antibody [EPR19781] (ab203786) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate
Lane 4 : Human spleen 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 1/10000 dilution

Predicted band size: 75 kDa
Observed band size: 75 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2,3 and 4: 30 seconds.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling MTHFR with ab203786 at 1/1000 (0.509 µg/mL) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human kidney. The section was incubated with ab203786 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution) for 20 minutes.

Immunohistochemical analysis of paraffin-embedded Human thyroid carcinoma tissue labeling MTHFR with ab203786 at 1/1000 (0.509 µg/mL) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human thyroid carcinoma. The section was incubated with ab203786 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution) for 20 minutes.

Lanes 1-3 : Anti-MTHFR antibody [EPR19781] (ab203786) at 1/1000 dilution
Lanes 4-5 : Anti-MTHFR antibody [EPR19781] (ab203786) at 1/5000 dilution

Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : HT-29 (Human colorectal adenocarcinoma cell line) whole cell lysate
Lane 3 : Human fetal liver lysate
Lane 4 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 5 : Mouse placenta 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: 75 kDa
Observed band size: 75 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-MTHFR antibody [EPR19781] (ab203786) at 1/1000 dilution

Lane 1 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 3 : Mouse kidney 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: 75 kDa
Observed band size: 75 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling MTHFR with ab203786 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on Jurkat cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab203786 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling MTHFR with ab203786 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on RAW 264.7 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab203786 at 1/100 dilution followed by ab150120  at 1/1000 dilution.

-ve control 2: ab7291  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

MTHFR was immunoprecipitated from 0.35mg of HT-29 (Human colorectal adenocarcinoma cell line) whole cell lysate with ab203786 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab203786 at 1/500 dilution.

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

Lane 1: HT-29 whole cell lysate, 10µg (Input).

Lane 2: ab203786 IP in HT-29 whole cell lysate.

Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab203786 in HT-29 whole cell lysate.

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

Exposure time: 10 seconds.