Anti-MIF antibody [EPR18149-128]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MIF antibody [EPR18149-128] (AB187064)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma cell line) cells labeling MIF with ab187064 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Neuro-2a cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MIF antibody [EPR18149-128] (AB187064)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized Neuro-2a (mouse neuroblastoma cell line) cell line labeling MIF with ab187064 at 1/500 (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MIF antibody [EPR18149-128] (AB187064)

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 MIF with ab187064 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on RAW 264.7 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MIF antibody [EPR18149-128] (AB187064)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell line labeling MIF with ab187064 at 1/500 (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-MIF antibody [EPR18149-128] (AB187064)

Exposure time : Lanes 1,2,3 and 4 : 10 seconds; Lanes 5 and 6 : 3 seconds; Lanes 7,8 and 9 : 1 second.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MIF antibody [EPR18149-128] (ab187064) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 20 µg

Lane 2:

Human fetal spleen lysate at 20 µg

Lane 3:

Mouse brain lysate at 20 µg

Lane 4:

Rat brain lysate at 20 µg

Lane 5:

J774A.1 (mouse reticulum cell sarcoma monocyte macrophage cell line) whole cell lysate at 20 µg

Lane 6:

Mouse kidney lysate at 20 µg

Lane 7:

Y79 (human retinoblastoma cell line) whole cell lysate at 20 µg

Lane 8:

Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 9:

Neuro-2a (mouse neuroblastoma cell line) whole cell lysate at 20 µg

Secondary

Lanes 1, 2, 7 and 8:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Lanes 3, 4, 5, 6 and 9:

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 1/100000 dilution

Predicted band size: 12 kDa

Observed band size: 13 kDa

true

• WB

Lab

Western blot - Anti-MIF antibody [EPR18149-128] (AB187064)

ab187064 was shown to specifically react with MIF in wild-type HAP1 cells as signal was lost in MIF knockout cells. Wild-type and MIF knockout samples were subjected to SDS-PAGE. ab187064 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

All lanes:

Western blot - Anti-MIF antibody [EPR18149-128] (ab187064) at 1/1000 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 20 µg/mL

Lane 2:

MIF knockout HAP1 whole cell lysate at 20 µg/mL

Lane 3:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg/mL

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg/mL

Predicted band size: 12 kDa

Observed band size: 13 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-MIF antibody [EPR18149-128] (AB187064)

Exposure time : Lane 1 : 5 seconds; Lane 2 : 1 second; Lane 3 : 30 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MIF antibody [EPR18149-128] (ab187064) at 1/5000 dilution

Lane 1:

NIH/3T3 (mouseembryo fibroblast cell line) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 3:

C6 (rat glial tumor cell line) 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 1/100000 dilution

Predicted band size: 12 kDa

Observed band size: 13 kDa

true