Anti-LAG-3 antibody [EPR20261] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

Tissue Microarrays stained for "Anti-LAG-3 antibody [EPR20261]" using "ab209236"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab209236 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

• Flow Cyt

Lab

Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with 5 μg/ml phytohaemagglutinin (PHA) for 72 hours (bottom), with ab209236 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10 μg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab209236 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 106 in 100 μl at 5.0 μg/ml (1/402)) for 30min on ice. The cells were simultaneously stained with CD3.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# ab209236).

Immunohistochemical analysis of paraffin-embedded human tonsil Hodgkin's lymphoma labeling LAG-3 with ab209236 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on immunocytes of the human Hodgkin's lymphoma [PMID : 11527700; PMID : 16757686].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

• Flow Cyt

Lab

Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

Flow cytometric analysis of Human peripheral blood mononuclear cells treated with 1 μg/mL PHA for 3 days cells with ab209236 at 1/50 dilution (right) compared with a rabbit monoclonal IgG isotype control (ab172730; left). ab150077 at 1/2000 dilution was used as the secondary antibody.

Only the CD3+ population are also positive for LAG-3. Gated on total viable cells.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

This ICC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# ab209236).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with GFP-tagged LAG3 expression construct or GFP only, labeling Lymphocyte Activation Gene 3 with ab209236 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 647) (ab150079) secondary antibody at 1/1000 dilution (green).

Confocal image showing positive staining on HEK-293T cells transfected with a GFP-tagged LAG3 expression construct.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 647) (ab150079) at 1/1000 dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

Flow cytometric analysis of HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with a GFP-tagged human LAG3 construct labeling LAG-3 with ab209236 at 1/500 dilution (right) compared with a rabbit monoclonal IgG isotype control (ab172730; left). Goat anti rabbit IgG (Alexa Fluor^® 647) ab150079 at 1/2000 dilution was used as the secondary antibody.

Note : Fresh cells without fixation and permeabilization were used to perform FC testing. Only GFP positive population results in LAG3 positive staining (Q2, right panel).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 with ab209236 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Sporadic cytoplasmic staining on immunocytes of human tonsil [PMID : 11527700; PMID : 16757686].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

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

• IP

Supplier Data

Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

LAG-3 was immunoprecipitated from 0.35 mg of HEK-293T (Human epithelial cell line from embryonic kidney) transfected with a GFP-tagged human LAG3 construct whole cell lysate with ab209236 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab209236 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate 10 μg (Input).

Lane 2 : ab209236 IP in HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab209236 in HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.

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

Exposure time : 1 second.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

All lanes:

Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] (<a href='/en-us/products/primary-antibodies/lag-3-antibody-epr20261-ab209236'>ab209236</a>)

Predicted band size: 57 kDa

false

• WB

Unknown

Western blot - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579)

This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# ab209236).

Primary loading control and concentration : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/20000 dilution

Secondary loading control and concentration : Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at 1/10000 dilution

Lanes 1-2 : Merged signal (red and green). Green – ab209236 observed at 54-70 kDa. Red - loading control ab8245 observed at 36 kDa.
ab209236 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® 800RCW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

The expression profile observed in Jurkat is consistent with the literature (PMID : 25108024).

Negative control : Jurkat (PMID : 25108024)

Observed MW : 54-70 kDa

All lanes:

Western blot - Anti-LAG-3 antibody [EPR20261] (<a href='/en-us/products/primary-antibodies/lag-3-antibody-epr20261-ab209236'>ab209236</a>) at 1/500 dilution

Lane 1:

HDLM-2 (Human Hodgkin lymphoma ) whole cell lysate at 20 µg

Lane 2:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 57 kDa

false