Anti-Mast Cell Tryptase antibody [AA1] (ab2378)

All lanes : Anti-Mast Cell Tryptase antibody [AA1] (ab2378) at 5 µg/ml

Lane 1 : Human lung normal tissue lysate - total protein (40 - 65 years) (ab30281)
Lane 2 : Human tonsil normal tissue lysate - total protein (ab29615)
Lane 3 : Human skin tissue lysate - total protein (ab30166)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 31 kDa
Observed band size: 35-37 kDa (why is the actual band size different from the predicted?)


Exposure time: 20 minutes

Mast Cell Tryptase contains an 18-residue signal sequence and a 12-residue activation peptide. The protein also has two potential glycosylation sites (Swissprot data). These post-translational modifications might explain the banding pattern observed. Abcam used 5% milk in TBS-T as a blocking agent for this blot. We found that this blocking agent removed more non-specific bands than BSA.

ab2378 staining Mast Cell Tryptase in Human Tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in a citric acid. Samples were incubated with primary antibody (1/7500 in TBS/BSA/azide) for 2 hours at 21°C. A Biotin-conjugated Goat anti-mouse IgG polyclonal (1/250) was used as the secondary antibody.

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ab2378 staining Mast Cell Tryptase in Human nasal polyp and tonsil tissue sections by Immunohistochemistry (resin-embedded sections). Tissue was fixed with acetone and inhibitors and embedded in GMA resin. Samples were incubated with primary antibody (1/2000 in Tris buffered saline) for 18 hours at 21°C. A Biotin-conjugated Rabbit anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.

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ab2378 staining Mast Cell Tryptase in Mouse RAW 264.7 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 10% Super block for 1 hour at 22°C. Samples were incubated with primary antibody (1/150 in PBS + 10% super block) for 14 hours at 4°C. An Alexa Fluor® 568-conjugated Goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody

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ab2378 staining Mast Cell Tryptase in Rat Lung tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. A Biotin-conjugated Goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody.

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ab2378 staining Mast Cell Tryptase in Mouse Lung (LPS inflammation) tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 4% M.O.M Ig blocking for 1 hour at 20°C; antigen retrieval was by heat mediation in a citric acid. Samples were incubated with primary antibody (1/200 in blocking buffer) for 16 hours at 4°C. A Biotin-conjugated Rabbit anti-mouse IgG polyclonal (1/100) was used as the secondary antibody.

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ab2378 staining formalin fixed paraffin-embedded human colon tissue sections cut at 1 micron.  The section was subjected to heat mediated antigen retrieval in Dako Target Retrieval Solution and permeabilized in Triton-X prior to blocking in 6% BSA for 2 hours at 24°C.  The primary anitbody was diluted 1/250 and incubated with the sample for 16 hours at 5°C.  The secondary antibody was ab7064 (diluted 1/2000) and the slide was counterstained with DAPI.

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ab2378 staining Mast Cell Tryptase in Human mast cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with methanol, permeabilized with Saponin 0.1% in PBS and blocked with 4% serum for 30 minutes at 25°C. Samples were incubated with primary antibody (1/100 in blocking buffer) for 14 hours at 4°C. A Texas Red^®-conjugated Goat anti-mouse polyclonal (1/100) was used as the secondary antibody.

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