Product datasheet

Anti-HLA A antibody [EP1395Y] ab52922
リコンビナント

15 Abreviews 96 References 画像数 15
製品の概要
製品名

Anti-HLA A antibody [EP1395Y]

製品の詳細

Rabbit monoclonal [EP1395Y] to HLA A

由来種

Rabbitアプリケーション

適用あり : Flow Cyt (Intra), WB, IP, IHC-P, ICC/IF

種交差性交差種 : Human

免疫原

Synthetic peptide within Human HLA A aa 50-150. The exact sequence is proprietary.
Database link: P04439

ポジティブ･コントロール

IHC-P: Human tonsil tissue. ICC/IF: MCF7 and Raji cells. WB: A431, Jurkat, THP-1, A549, HL-60
and Raji cell lysates. IP: THP-1 and A549 cell lysates. Flow Cyt (intra): Raji cells.

特記事項This product is a recombinant monoclonal antibody, which offers several advantages including:- High batch-to-batch consistency and reproducibility- Improved sensitivity and specificity- Long-term security of supply- Animal-free production
For more information see here.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit
monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

製品の特性
製品の状態

Liquid

保存方法

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C.
Avoid freeze / thaw cycle.

バッファー

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA

精製度

Protein A purified

ポリ /モノモノクローナル

クローン名

EP1395Y
1

アイソタイプ

IgG

アプリケーション

The Abpromise guarantee

Abpromise保証は、 次のテスト済みアプリケーションにおけるab52922の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション

特記事項

Abreviews

Flow Cyt (Intra)

WB

1/10 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as anisotype control with this antibody.

(4)

1/2000. Predicted molecular weight: 41 kDa.

For unpurified use at 1/10000 - 1/50000.
IPIHC-P

1/20. For unpurified use at 1/30.

(5)

1/100. Perform heat mediated antigen retrieval with Tris/EDTA
buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified use at 1/250 - 1/500.
See IHC antigen retrieval protocols.

ICC/IF(1)

1/100. For unpurified use at 1/250 - 1/500.

ターゲット情報関連性

HLA-A belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer
consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchoredin the membrane. Class I molecules play a central role in the immune system by presenting
peptides derived from the endoplasmic reticulum lumen. They are expressed in nearly all cells.
The heavy chain is approximately 45 kDa and its gene contains 8 exons. Exon 1 encodes theleader peptide, exons 2 and 3 encode the alpha1 and alpha2 domains, which both bind thepeptide, exon 4 encodes the alpha3 domain, exon 5 encodes the transmembrane region, andexons 6 and 7 encode the cytoplasmic tail. Polymorphisms within exon 2 and exon 3 are
responsible for the peptide binding specificity of each class one molecule. Typing for thesepolymorphisms is routinely done for bone marrow and kidney transplantation. Hundreds of HLA-A
alleles have been described.

画像2

All lanes : Anti-HLA A antibody [EP1395Y] (ab52922) at 1/10000
dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : HLA-A knockout A549 cell lysate
Lane 3 : Wild-type A431 cell lysate
Lane 4 : HLA-A knockout A431 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Western blot - Anti-HLA A antibody [EP1395Y](ab52922)

Predicted band size: 41 kDa
Observed band size: 41 kDa

Anti-HLA-A antibody [EP1395Y] (ab52922) staining at 1/10000
dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A](ab7291) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab52922 was shown to bind specifically to HLA-A.
A band was observed at 41 kDa in wild-type A549 cell lysates withno signal observed at this size in HLA-A knockout cell line. Togenerate this image, wild-type and HLA-A knockout A549 celllysates were analysed. First, samples were run on an SDS-PAGE
gel then transferred onto a nitrocellulose membrane. Membranes
were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before
incubation with primary antibodies overnight at 4 °C. Blots were
washed four times in TBS-T, incubated with secondary antibodies
for 1 h at room temperature, washed again four times then imaged.
Secondary antibodies used were Goat anti-Rabbit IgG H&L
800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embeddedsections) analysis of human tonsil tissue labelling HLA A withpurified ab52922 at 1/100. Heat mediated antigen retrieval wasperformed using EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG
H&L (HRP) was used as the secondary antibody (1/500). Negative
control using PBS instead of primary antibody. Counterstained withhematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-HLA A antibody[EP1395Y] (ab52922)

All lanes : Anti-HLA A antibody [EP1395Y] (ab52922) at 1/10000
dilution

Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line)
whole cell lysate

Lane 2 : HLA A knockout A-431 (Human epidermoid carcinoma
cell line) whole cell lysate

Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheralblood) whole cell lysate

Western blot - Anti-HLA A antibody [EP1395Y]Lysates/proteins at 20 µg per lane.

(ab52922)

Performed under reducing conditions.

Predicted band size: 41 kDa
Observed band size: 40 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab52922
observed at 40 kDa. Red - loading control, ab7291 (Mouse antiAlpha Tubulin [DM1A] observed at 55kDa.
4ab52922 was shown to react with HLA-A in A431 wild-type cells in
Western blot. Loss of signal was observed when HLA-A knockout
sample was used. A431 wild-type and HLA-A knockout cell lysateswere subjected to SDS-PAGE. Membranes were blocked in 3%
Milk in TBS-T (0.1% Tween®) before incubation with ab52922 andab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1in 10000 dilution and a 1 in 20000 dilution respectively. Blots weredeveloped with Goat anti-Rabbit IgG H&L (IRDye® 800CW)
preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®
680RD) preabsorbed (ab216776) secondary antibodies at 1 in
20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing Raji cells stained with ab52922 (redline). The cells were fixed with 4% paraformaldehyde (10 min) andthen permeabilized with 0.1% PBS-Tween for 20 min. The cells
were then incubated in 1x PBS / 10% normal goat serum / 0.3M
glycine to block non-specific protein-protein interactions followed by
the antibody (ab52922, 1/100) for 30 min at 22°C. The secondary
antibody used was DyLight® 488 goat anti-rabbit IgG (H+L)(ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control
Flow Cytometry (Intracellular) - Anti-HLA A antibodyantibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells)[EP1395Y] (ab52922)

used under the same conditions. Acquisition of >5,000 events wasperformed. This antibody gave a positive signal in Raji cells fixedwith 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for
20 min used under the same conditions.

Immunocytochemistry/Immunofluorescence analysis of Raji (humanBurkitt's lymphoma) cells labelling HLA A with purified ab52922 at
1/100. Cells were fixed with 4% paraformaldehyde andpermeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor®
488-conjugated goat anti-rabbit IgG (1/1000) was used as thesecondary antibody. DAPI (blue) was used as the nuclear
counterstain. ab7291, a mouse anti-tubulin (1/1000) andab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG(1/1000) were also used.
Control 1: primary antibody (1/100) and secondary antibody,ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG
Immunocytochemistry/ Immunofluorescence - AntiHLA A antibody [EP1395Y] (ab52922)(1/500).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, anAlexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

5All lanes : Anti-HLA A antibody [EP1395Y] (ab52922) at 1/5000
dilution (purified)

Lane 1 : THP-1 cell lysate at 20 µg
Lane 2 : A549 cell lysate at 1/20 dilution
SecondaryAll lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000
dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 41 kDa
Western blot - Anti-HLA A antibody [EP1395Y](ab52922)

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

ab52922 (purified) at 1/20 immunoprecipitating HLA A in THP-1
whole cell lysate. 10 ug of cell lysate was present in the input. For
western blotting, a HRP-conjugated Veriblot for IP Detection
Reagent (ab131366) (1/10,000) was used for detection. A rabbit
monoclonal IgG (ab172730) was used intead of ab128913 as anegative control (Lane 3).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

Immunoprecipitation - Anti-HLA A antibody[EP1395Y] (ab52922)

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Intracellular Flow Cytometry analysis ofRaji cells labelling HLA Awith
purified ab52922 at 1/40 (red). Cells were fixed with 2%paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500)
was used as the secondary antibody. Black - Isotype control, rabbit
monoclonal IgG. Blue - Unlabelled control, cells without incubation
with primary and secondary antibodies.

Flow Cytometry (Intracellular) - Anti-HLA A antibody[EP1395Y] (ab52922)

ICC/IF image of unpurified ab52922 stained MCF7 cells. The cells
were 4% formaldehyde fixed (10 min) and then incubated in
1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBSTween for 1h to permeabilise the cells and block non-specificprotein-protein interactions. The cells were then incubated with theantibody (ab52922, 5µg/ml) overnight at +4°C. The secondary
antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L)
used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used
to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI
was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Immunocytochemistry/ Immunofluorescence - AntiHLA A antibody [EP1395Y] (ab52922)

Anti-HLA A antibody [EP1395Y] (ab52922) at 1/2000 dilution(purified) + HL-60 cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution(Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 41 kDa

Western blot - Anti-HLA A antibody [EP1395Y]

Blocking buffer and concentration: 5% NFDM/TBST.

(ab52922)

Diluting buffer and concentration: 5% NFDM /TBST.

7

Anti-HLA A antibody [EP1395Y] (ab52922) at 1/10000 dilution +Raji cell lysate at 10 µg

Secondary
Goat anti rabbit IgG HRP conjugated at 1/2000 dilution

Predicted band size: 41 kDa
Observed band size: 41 kDa

Western blot - Anti-HLA A antibody [EP1395Y](ab52922)

ab52922 staining HLA A in Human tonsil tissue sections byImmunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffinembedded sections). Tissue was fixed with formaldehyde andblocked with 3% H2O2 for 10 minutes at 25°C; antigen retrieval wasby heat mediation in a citrate buffer, pH 6.0 . Samples wereincubated with primary antibody (1/3000) for 20 minutes at 25°C.
An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal wasused as the secondary antibody.

Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-HLA A antibody[EP1395Y] (ab52922)
This image is courtesy of an anonymous AbreviewAb52922 at 1/250 dilution staining human tonsil; paraffinembedded.

Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-HLA A antibody[EP1395Y] (ab52922)

8

ab52922 (purified) at 1/20 immunoprecipitating HLA A in A549
whole cell lysate. 10 ug of cell lysate was present in the input. For
western blotting, a HRP-conjugated Veriblot for IP Detection
Reagent (ab131366) (1/10,000) was used for detection. A rabbit
monoclonal IgG (ab172730) was used intead of ab128913 as anegative control (Lane 3).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

Immunoprecipitation - Anti-HLA A antibody[EP1395Y] (ab52922)

Anti-HLA A antibody [EP1395Y] (ab52922)

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