Anti-MHC Class I H2-Kb antibody [MJF-D30006-520]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (AB327060)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized EL4 (mouse lymphoma T lymphocyte) RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling MHC Class I H2‑Kb with ab327060 at 1/500 (1.012 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in EL4 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Low expression : RAW264.7(PMID : 35965334) Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab327060 at 1/200 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• WB

Lab

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (AB327060)

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

This antibody does not cross-react with mouse PH-2D, H2-Q10, H2-Q8, H2-Q7 and H2-L.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1 : 100000 (15Kda); Anti-6X His tag antibody [EPR20547]-CHIP Grade (ab213204) (1 : 5000)(37-50Kda).

All lanes:

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (ab327060) at 1/1000 dilution

Lane 1:

293T cells transfected with a mouse K-B expression vector containing a his tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with an empty vector containing a his-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse PH-2D expression vector containing a his tag, whole cell lysate at 80 µg

Lane 4:

293T cells transfected with a mouse H2-Q10 expression vector containing a his tag, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a mouse H2-Q8 expression vector containing a his tag, whole cell lysate at 20 µg

Lane 6:

293T cells transfected with a mouse H2-Q7 expression vector containing a his tag, whole cell lysate at 20 µg

Lane 7:

293T cells transfected with a mouse H2-L expression vector containing a 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: 37-50 kDa,15 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (AB327060)

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

This antibody does not cross-react with mouse D-P, D-K, D-B and D-D.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1 : 100000 (15Kda); Anti-6X His tag antibody [EPR20547]-CHIP Grade (ab213204) (1 : 5000)(37-50Kda).

All lanes:

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (ab327060) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a his-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a mouse K-B expression vector containing a his tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse D-P expression vector containing a his tag, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a mouse D-K expression vector containing a his tag, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a mouse D-B expression vector containing a his tag, whole cell lysate at 20 µg

Lane 6:

293T cells transfected with a mouse D-D expression vector containing a 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: 37-50 kDa,15 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (AB327060)

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

Low expression : Raw 264.7(PMID : 35965334), NIH/3T3, MEF.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (ab327060) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 50 µg

Lane 2:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 50 µg

Lane 3:

MEF (mouse embryo fibroblast) whole cell lysate at 50 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 50 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: 37-50 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (AB327060)

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

This antibody does not cross-react with mouse K-D, K-K, K-Q, K-W28 and alpha chain.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1 : 100000 (15Kda); Anti-6X His tag antibody [EPR20547]-CHIP Grade (ab213204) (1 : 5000)(37-50Kda).

All lanes:

Western blot - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (ab327060) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a his-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a mouse K-B expression vector containing a his tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse K-D expression vector containing a his tag, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a mouse K-K expression vector containing a his tag, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a mouse K-Q expression vector containing a his tag, whole cell lysate at 20 µg

Lane 6:

293T cells transfected with a mouse K-W28 expression vector containing a his tag, whole cell lysate at 20 µg

Lane 7:

293T cells transfected with a mouse alpha chain expression vector containing a 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: 37-50 kDa,15 kDa

false

Exposure time: 15s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MHC Class I H2-Kb antibody [MJF-D30006-520] (AB327060)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells transfected with a H-2 class I histocompatibility antigen(D-B alpha chain) protein expression vector containing a Myc-His-tag® 293T cells transfected with a H-2 class I histocompatibility antigen(D-D alpha chain) protein expression vector containing a Myc-His-tag® 293T cells transfected with a H-2 class I histocompatibility antigen(alpha chain) protein expression vector containing a Myc-His-tag® 293T cells transfected with a H-2 class I histocompatibility antigen(D-P alpha chain) protein expression vector containing a Myc-His-tag® 293T cells transfected with a H-2 class I histocompatibility antigen(D-K alpha chain) protein expression vector containing a Myc-His-tag® cells labelling MHC Class I H2‑Kb with ab327060 at 1/500 (1.012 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing positive staining in 293T cells transfected with a H-2 class I histocompatibility antigen(K-B alpha chain) protein expression vector containing a Myc-His-tag® (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). Panel G : 293T cells transfected with a H-2 class I histocompatibility antigen(D-B alpha chain) protein expression vector containing a Myc-His-tag® Panel H : 293T cells transfected with a H-2 class I histocompatibility antigen(D-D alpha chain) protein expression vector containing a Myc-His-tag® Panel I : 293T cells transfected with a H-2 class I histocompatibility antigen(alpha chain) protein expression vector containing a Myc-His-tag® Panel J : 293T cells transfected with a H-2 class I histocompatibility antigen(D-P alpha chain) protein expression vector containing a Myc-His-tag® Panel K : 293T cells transfected with a H-2 class I histocompatibility antigen(D-K alpha chain) protein expression vector containing a Myc-His-tag®

ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/100 dilution (Magenta). 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 dilution.