All lanes : Anti-IL37 antibody [EPR24011-161] (ab278499) at 1/5000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lane 2 : THP-1 treated with 100nM PMA for 16 hours. And then replace PMA with /ml LPS(Lipopolysaccharide) for 4 hours, and then 1/ml Brefeldin A was added for the last 3 hours, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 24 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

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

IL 37 form homodimer through non-disulfide bonds. The molecular weight observed is consistent with what have been described in literature (PMID:20935647, 28783685).

PMA and LPS treatment increase the expression of IL 37 (PMID:20935647).

Exposure time: 48 seconds

 

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling IL37 with ab278499 at 1/5000 (0.21 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human testis (PMID:11145836). The section was incubated with ab278499 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 cells labelling IL37 with ab278499 at 1/250 (2.272 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing strong cytoplasmic and weak nuclear staining in THP-1 cells treated with phorbol 12-myristate 13-acetate (100nM) for 16 hours, and then with Lipopolysaccharide (100 ng/ml) for 4 hours and co-treated with brefeldin A (1ug/ml) for another 3 hours. PMA and LPS treatment increase the expression of IL 37 (PMID:20935647) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

IL37 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) treated with 100nM PMA for 16 hours. And then replace PMA with 100 ng/ml LPS (Lipopolysaccharide) for 4 hours, and then 1 ug/ml Brefeldin A was added for the last 3 hours, whole cell lysate 10 ug with ab278499 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab278499 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (human monocytic leukemia monocyte) treated with 100nM PMA for 16 hours. And then replace PMA with 100 ng/ml LPS(Lipopolysaccharide) for 4 hours, and then 1 ug/ml Brefeldin A was added for the last 3 hours, whole cell lysate 10 ug

Lane 2: ab278499 IP in THP-1 treated with 100nM PMA for 16 hours. And then replace PMA with 100 ng/ml LPS(Lipopolysaccharide) for 4 hours, and then 1 ug/ml Brefeldin A was added for the last 3 hours whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab278499 in THP-1 treated with 100nM PMA for 16 hours. And then replace PMA with 100 ng/ml LPS(Lipopolysaccharide) for 4 hours, and then 1 ug/ml Brefeldin A was added for the last 3 hours whole cell lysate

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

Exposure time: 15 seconds

 

Anti-IL37 antibody [EPR24011-161] (ab278499) at 1/5000 dilution + Human IL-37 recombinant protein (aa46-218), 10 ng

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 24 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

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

Exposure time: 10 seconds

 

Immunohistochemical analysis of paraffin-embedded Human skin tissue labeling IL37 with ab278499 at 1/5000 (0.21 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on granular layer of human skin (PMID:24441097). The section was incubated with ab278499 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Anti-IL37 antibody [EPR24011-161] (ab278499) at 1/1000 dilution + Human testis tissue lysate at 20 µg

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution (VeriBlot for IP secondary antibody(HRP))

Predicted band size: 24 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

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

IL 37 form homodimer through non-disulfide bonds. The molecular weight observed is consistent with what have been described in literature (PMID:20935647, 28783685).

Exposure time: 37 seconds

 

Immunohistochemical analysis of paraffin-embedded Human psoriasic skin tissue labeling IL37 with ab278499 at 1/5000 (0.21 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining on human psoriasic skin (PMID:24441097). The section was incubated with ab278499 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.