Astana Biomed Group, an authorized Abcam distributor in Central Asia

Inhibitory Immune Checkpoint panel 2 - Human WB (ab278173)

Overview

  • Product name

    Inhibitory Immune Checkpoint panel 2 - Human WB

  • Species reactivity

    Reacts with: Human

  • Product overview

    Inhibitory Immune Checkpoint panel 2 - Human WB ab278173 contains multiple trial-sized versions of anti-human antibody clones against CD47, SIRP alpha, NKG2A, galectin 9/Gal-9, Indoleamine 2, 3-dioxygenase, 2B4 and CD276. For guidelines on how to use each antibody within the panel, please consult the individual datasheet for each antibody.specifically selected for high performance in various applications.  They are provided as a sampler panel to allow you to easily evaluate each antibody.


    Panel contains:


    - Rabbit monoclonal [EPR21794] to CD47 (20 μL) ab218810


    - Rabbit monoclonal [EPR16264] to SIRP alpha (20 μL) ab191419


    - Rabbit monoclonal [EPR23620-39] to NKG2A (20 μL) ab252437


    - Rabbit monoclonal [EPR22214] to galectin 9/Gal-9 (20 μL) ab227046 


    - Rabbit monoclonal [EPR20374] to Indoleamine 2, 3-dioxygenase (20 μL) ab211017


    - Rabbit monoclonal [EPR23692-33] to 2B4 (20 μL) ab256370


    - Rabbit monoclonal [SP206] to CD276 (20 μL) ab227670

  • Notes

    Explore our range of antibody sample panels designed to provide you with a variety of trial-size antibodies in a convenient and cost-effective format.

    Directly conjugated versions of our antibodies are available and ready to use for multicolor flow cytometry or immunocytochemistry analysis. Please refer to the ‘Associated products’ section below.

    Carrier-free formulations of our recombinant antibodies are also available for easy conjugation to labels of your choice and for multiplex applications. Please refer to the ‘Associated products’ section below.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

Images

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

     

    All lanes: Anti-CD276 antibody [SP206] (ab227670) at 1/1000 dilution.

    Lane 1: Wild-type HEK293T cell lysate, 20 ug

    Lane 2: CD276 knockout HEK293T cell lysate, 20 ug

    Lane 3:  LNCaP cell lysate, 20 ug

    Lane 4: Raji cell lysate, 20 ug

    Secondary (all lanes): ab216773 at 1/10000 dilution.

    Predicted MW: 57kDa.

    Observed MW: 90-110kDa

    Lanes 1-4: Merged signal (red and green). Green - ab227670 observed at 90-110 kDa. Red - loading control ab8245 observed at 36 kDa.

    ab227670 Anti-CD276 antibody [SP206] was shown to specifically react with CD276 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266658 (knockout cell lysate ab257097) was used. Wild-type and CD276 knockout samples were subjected to SDS-PAGE. ab227670 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-CD276 antibody [SP206] (ab227670) at 1/400 dilution.

    Lane 1: HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate

    Predicted MW: 57 kDa.

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-2B4 antibody [EPR23692-33] (ab256370) at 1/1000 dilution.

    Lane 1: Human spleen tissue lysate, 20 ug

    Secondary (all lanes): ab131366 at 1/1000 dilution.

    Predicted MW: 42 kDa.

    Observed MW: 70 kDa

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

    This blot was developed using a higher sensitivity ECL substrate.

    Exposure time: 48 seconds.

     

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-2B4 antibody [EPR23692-33] (ab256370) at 1/1000 dilution.

    Lane 1: KG-1a (human bone marrow myeloblast) whole cell lysate, 10 ug

    Secondary (all lanes): ab131366 at 1/5000 dilution.

    Predicted MW: 42 kDa.

    Observed MW: 70 kDa

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

    This blot was developed using a higher sensitivity ECL substrate.

    Exposure time: 24 seconds.

     

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-2B4 antibody [EPR23692-33] (ab256370) at 1/1000 dilution.

    Lane 1: NK-92 (human malignant non-Hodgkins lymphoma natural killer cell) whole cell lysate, 20 ug

    Lane 2: NK-92 whole cell lysate treated with PNGase F, 20 ug

    Lane 3: A549 (human lung carcinoma epithelial cell) whole cell lysate, 20 ug.

    Lane 4: THP-1 (human monocytic leukemia monocyte) whole cell lysate, 20 ug

    Secondary (all lanes): ab131366 at 1/1000 dilution.

    Predicted MW: 42 kDa.

    Observed MW: 37, 42, 70-100 kDa

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

    The molecular weight observed is consistent with what has been described in the literature (PMID: 19499526, 11099315, 16177062, 28126968).

    Negative control: A549 (PMID: 23192659 and Human Protein Atlas).

    Exposure time: Lanes 1-2: 37 seconds; Lanes 3-4: 125 seconds.

     

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 dilution.

    Lane 1: Human ovary cancer lysate, 20 ug

    Lane 2: Human placenta lysate, 20 ug

    Lane 3: Human tonsil lysate, 20 ug

    Lane 4: SK-OV-3 (Human ovarian cancer cell line) whole cell lysate, 10 ug

    Secondary (all lanes): ab97051 at 1/100000 dilution.

    Predicted MW:  45kDa.

    Observed MW: 45kDa

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1-3: 3 minutes; Lane 4: 15 seconds.

     

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 dilution.

    Lane 1: Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate, 10 ug

    Lane 2: HeLa whole cell lysate treated with 50ng/ml Interferon-gamma (IFN-gamma, ab51240) for 16 hours, 10 ug

    Secondary (all lanes): ab97051 at 1/100000 dilution.

    Predicted MW:  45kDa.

    Observed MW: 45kDa

    Blocking/Dilution buffer: 5% NFDM/TBST.

    The level of Indoleamine 2, 3-dioxygenase expression can be induced by IFN-γ treatment (PMID 16368976).

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/1000 dilution.

    Lane 1: THP-1 (human monocytic leukemia monocyte), whole cell lysate, 20 ug

    Lane 2: HEK-293T (human embryonic kidney epithelial cell), whole cell lysate, 20 ug

    Secondary (all lanes): ab97051 at 1/20000 dilution.

    Predicted MW: 40 kDa.

    Observed MW: 34-39kDa.

     

    Blocking/Dilution buffer: 5% NFDM/TBST.

    The expression of the isoforms observed is consistent with what has been described in the literature (PMID 11886844, PMID: 22805533).

    Degraded fragments (13-15 kDa) has also been described (PMID: 15811318).

    Negative control: HEK-293T (PMID: 24333696).

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/1000 dilution.

     

    Lane 1:  U937 (human histiocytic lymphoma monocyte), whole cell lysate, 20 ug

    Lane 2:  HL-60 (human Acute Promyelocytic Leukemia promyeloblast), whole cell lysate, 20 ug

    Lane 3:      MOLT-4 (human lymphoblastic leukemia T lymphoblast), whole cell lysate, 20 ug

    Lane 4:     PANC-1 (human pancreatic epithelioid carcinoma epithelial cell), whole cell lysate, 20 ug

    Secondary (all lanes): ab97051 at 1/20000 dilution.

    Predicted MW: 40 kDa.

    Observed MW: 34-39kDa. 13-15kDa - degradation product

    Blocking/Dilution buffer: 5% NFDM/TBST.

    The expression of the isoforms observed is consistent with what has been described in the literature (PMID 11886844, PMID: 22805533).

    Degraded fragments (13-15 kDa) have also been descried (PMID: 15811318).

    Negative control: PANC-1.

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-NKG2A antibody [EPR23620-39] (ab252437) at 1/1000 dilution.

    Lane 1: NK-92 (human malignant non-Hodgkin's lymphoma natural killer cell), whole cell lysate, 20 ug

    Secondary (all lanes): ab97051 at 1/100000 dilution.

    Predicted MW: 26 kDa.

    Observed MW: 34-38 kDa

     

    Blocking and dilution buffer: 5% NFDM/TBST.

    Expossure time: 3 minutes.

    This blot was developed using a higher sensitivity ECL substrate.

    Multiple bands are likely due to isoforms and glycosylation. The molecular profile/weight observed is consistent with what has been described in the literature (PMID: 9034158).

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-NKG2A antibody [EPR23620-39] (ab252437) at 1/1000 dilution.

    Lane 1: HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate, 10 ug.

    Lane 2: HEK-293T transfected with NKG2-A (WT) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug

    Lane 3: HEK-293T transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate, 10 ug

    Lane 4: HEK-293T transfected with NKG2-C (WT) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug

    Lane 5: HEK-293T transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate, 10 ug

    Lane 6: HEK-293T transfected with NKG2-E (WT) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug

    Secondary (all lanes): ab97051 at 1/100000 dilution.

    Predicted MW: 26 kDa.

    Blocking and dilution buffer: 5% NFDM/TBST.

    Exposure time: 3 minutes.

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-SIRP alpha antibody [EPR16264] (ab191419) at 1/1000 dilution.

    Lane 1: Wild-type HAP1 whole cell lysate, 20 ug.

    Lane 2: SIRPA knockout HAP1 whole cell lysate, 20 ug

    Lane 3: THP1 whole cell lysate, 20 ug.

    Lane 4: Jurkat whole cell lysate (negative control), 20 ug.

    Lanes 1 - 4: Merged signal (red and green). Green - ab191419 observed at 55 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab191419 was shown to recognize SIRP alpha in wild-type HAP1 cells as signal was lost at the expected MW in SIRPA knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SIRPA knockout samples were subjected to SDS-PAGE. Ab191419 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-SIRP alpha antibody [EPR16264] (ab191419) at 1/5000 dilution.

    Lane 1: THP1 cell lysate, 20 ug.

    Lane 2: SW480 cell lysate, 20 ug

    Lane 3: Human fetal brain lysate, 20 ug.

    Secondary (all lanes): Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated, at 1/1000 dilution.

    Predicted MW: 55kDa.

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-CD47 antibody [EPR21794] (ab218810) at 1/500 dilution.

    Lane 1: Wild-type HEK293T cell lysate, 20 ug. 


    Lane 2:     CD47 knockout HEK293T cell lysate, 20 ug.

    Lane 3:      Jurkat cell lysate, 20 ug.

    Lane 4:      HepG2 cell lysate, 20 ug.

    Secondary (all lanes):  ab216773 at 1/10000 dilution.

    Predicted MW: 35 kDa.

    Observed MW: 47-52kDa.

     

    Lanes 1-4: Merged signal (red and green). Green - ab218810 observed at 47-52 kDa. Red - loading control ab8245 observed at 36 kDa.

    ab218810 Anti-CD47 antibody [EPR21794] was shown to specifically react with CD47 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266324 (knockout cell lysate ab257220) was used. Wild-type and CD47 knockout samples were subjected to SDS-PAGE. ab218810 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

  • Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)
    Western blot - Inhibitory immune checkpoint panel 2 - human WB (ab278173)

    All lanes: Anti-CD47 antibody [EPR21794] (ab218810) at 1/5000 dilution.

    Lane 1: Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate 20 ug.

    Lane 2: U937 (human  histiocytic lymphoma cell line) whole cell lysate, 20 ug.

    Lane 3: A549 (human lung carcinoma cell line) whole cell lysate, 20 ug. 

    Lane 4: U-2 OS (human bone osteosarcoma epithelial cell line) whole cell lysate, 20 ug

    Lane 5: Human brain lysate, 10ug

    Secondary (all lanes): ab97051 at 1/100000 dilution.

    Predicted MW: 35 kDa.

    All lanes: Anti-PD-L1 antibody [73-10] (ab228415) at 1/1000 dilution.

    Lane 1: NCI-H1975 (human non-small cell lung cancer cell line), whole cell lysate, 20 ug.

    Lane 2: Human placenta, 20 ug.

    Lane 3: Human thymus, 20 ug. 

    Secondary (all lanes): ab97051 at 1/50000 dilution.

    Predicted MW: 33 kDa.

    Observed MW: 47-52 kDa. The molecular mass observed is consistent with the literature (PMID 12393467, PMID 11034562).

    Exposure time: Lane 1: 26 seconds; Lane 2: 70 seconds; Lanes 3-4: 3 minutes; Lane 5: 3 seconds.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    We recommend that samples are not boiled after adding loading buffer as this may cause protein aggregates. 

     

     

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