Anti-fetal hemoglobin antibody [EPR23381-254]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-fetal hemoglobin antibody [EPR23381-254] (AB283313)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized K-562 cells labelling fetal hemoglobin with ab283313 at 1/50 (10.92 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing cytoplasmic staining in K-562 cells and no staining in HeLa cells.

Negative control : HeLa (PMID : 17910885) is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-fetal hemoglobin antibody [EPR23381-254] (AB283313)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell, Left) / K-562 (Human chronic myelogenous leukemia lymphoblast, Right) cells labelling fetal hemoglobin with ab283313 at 1/500 dilution (0.1ug) (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.

Negative control : HeLa (PMID : 17910885).

• IP

Supplier Data

Immunoprecipitation - Anti-fetal hemoglobin antibody [EPR23381-254] (AB283313)

fetal hemoglobin was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug with ab283313 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283313 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug

Lane 2 : ab283313 IP in K-562 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283313 in K-562 whole cell lysate

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

Exposure time : 32 seconds

All lanes:

Immunoprecipitation - Anti-fetal hemoglobin antibody [EPR23381-254] (ab283313)

Predicted band size: 16 kDa

Observed band size: 14 kDa

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• WB

Lab

Western blot - Anti-fetal hemoglobin antibody [EPR23381-254] (AB283313)

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

Negative control : HeLa (PMID : 17910885).

Extra bands around 40 kDa and 150 kDa can be observed in some adult tissue lysates.

Exposure time : 59 seconds

All lanes:

Western blot - Anti-fetal hemoglobin antibody [EPR23381-254] (ab283313) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HEL (human erythroleukemia erythroblast) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 16 kDa

Observed band size: 14 kDa

false

• WB

Lab

Western blot - Anti-fetal hemoglobin antibody [EPR23381-254] (AB283313)

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

Exposure time : 3 seconds

These rec proteins were made in house.
Samples loaded are extracts of E.coli expressing
HBG2, HBG1, HBE1, HBB or HBD.

All lanes:

Western blot - Anti-fetal hemoglobin antibody [EPR23381-254] (ab283313) at 1/1000 dilution

Lane 1:

His-tagged human HBG2 recombinant protein (aa2-147) at 0.01 µg

Lane 2:

His-tagged human HBG1 recombinant protein (aa2-147) at 0.01 µg

Lane 3:

His-tagged human HBE1 recombinant protein (aa3-147) at 0.01 µg

Lane 4:

His-tagged human HBB recombinant protein (aa3-147) at 0.01 µg

Lane 5:

His-tagged human HBD recombinant protein (aa3-147) at 0.01 µg

Secondary

Lanes 1 - 5:

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

Lanes 1 - 5:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Predicted band size: 16 kDa

Observed band size: 15 kDa

false