Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (AB259808)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney) cells labelling KIR3DL1+KIR3DS1+KIR2DS2+KIR2DS4 with ab259808 at 1/50 dilution (10.2 ug/ml), followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) antibody at 1/500 dilution (Green). Confocal image showing cytoplasmic staining in 293T cells transfected with myc-tagged KIR3DL1 expression vector is observed.

Myc-Tag Mouse mAb (Alexa Fluor^® 488) was used to counterstain myc-tag at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at 1/500 dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (AB259808)

Flow cytometric analysis of Human peripheral blood mononuclear cell (PBMC) cells labelling KIR3DL1+KIR3DS1+KIR2DS2+KIR2DS4 with ab259808 at 1/500 dilution (0.1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. Goat anti rabbit IgG (Alexa Fluor^® 647, ab150079) at 1/2000 dilution was used as the secondary antibody. Cells were stained with rabbit IgG or ab259808. Then stained with anti-CD56 conjugated to BV421. Gated on viable cells.

• IP

Supplier Data

Immunoprecipitation - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (AB259808)

KIR3DL1+KIR3DS1+KIR2DS2+KIR2DS4 was immunoprecipitated from 0.35 mg 293T transfected with human KIR3DL1 expression vector containing a myc-His-tag^®, whole cell lysate with ab259808 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259808 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T (human embryonic kidney) transfected with human KIR3DL1 expression vector containing a myc-His-tag^®, whole cell lysate 10 ug

Lane 2 : ab259808 IP in 293T transfected with human KIR3DL1 expression vector containing a myc-His-tag^®, whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab259808 in 293T transfected with human KIR3DL1 expression vector containing a myc-His-tag^®, whole cell lysate

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

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (ab259808)

Observed band size: 60-80 kDa

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• WB

Lab

Western blot - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (AB259808)

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

The expression pattern & observed MW are consistent with what has been described in the literature (PMID : 17911614).

Negative control : human tonsil (PMID : 14734722）

Exposure time : 3 minutes

All lanes:

Western blot - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (ab259808) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 60 µg

Lane 2:

Human tonsil tissue lysate at 60 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 60-80 kDa

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• WB

Lab

Western blot - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (AB259808)

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

This antibody has high cross-reactivity with human KIR2DS4, and low with human KIR2DS2. This antibody does not recognize human KIR3DL3.

All of the rec proteins were made in-house. KIR3DL1 and KIR2DS2 were expressed from HEK-293 expression systems. KIR3DL3 and KIR2DS4 were expressed from E.coli expression systems.

Exposure time : 15 seconds

All lanes:

Western blot - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (ab259808) at 1/1000 dilution

Lane 1:

His-tagged human KIR3DL1 recombinant protein at 0.01 µg

Lane 2:

His-tagged human KIR3DL3 recombinant protein at 0.01 µg

Lane 3:

His-tagged human KIR2DS4 recombinant protein at 0.01 µg

Lane 4:

LIF/His-tagged human KIR2DS2 recombinant protein at 0.01 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

Observed band size: 33-87 kDa

false

• WB

Lab

Western blot - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (AB259808)

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

This antibody can recognize human KIR3DS1 but can not recognize human KIR3DL2.

Exposure time : Lane 1-4 : 3 minutes Lane 5-6 : 59 seconds

All lanes:

Western blot - Anti-KIR3DL1 + KIR2DS4 + KIR2DS2 + KIR3DS1 antibody [EPR24357-139] (ab259808) at 1/1000 dilution

Lanes 1, 3 and 5:

293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

293T transfected with human KIR3DL1 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 4:

293T transfected with human KIR3DL2 expression vector containing a myc-His-tag®, whole cell lysate 20 at 20 µg

Lane 6:

293T transfected with human KIR3DS1 expression vector containing a myc-His-tag®, 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

Observed band size: 45-70 kDa

false