Anti-CD3 antibody (ab16044)
Key features and details
- Rabbit polyclonal to CD3
- Suitable for: IP, WB
- Reacts with: Mouse, Rat, Human
- Isotype: IgG
Overview
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Product name
Anti-CD3 antibody
See all CD3 primary antibodies -
Description
Rabbit polyclonal to CD3 -
Host species
Rabbit -
Tested applications
Suitable for: IP, WBmore details
Unsuitable for: IHC -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide conjugated to KLH derived from within residues 150 to the C-terminus of Human CD3.
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Positive control
- Recombinant Human CD3 epsilon protein (ab114153) can be used as a positive control in WB. This antibody gave a positive signal in Jurkat whole cells and thymus tissue from Mouse and Rat.
Images
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All lanes : Anti-CD3 antibody (ab16044) at 1 µg/ml
Lane 1 : THP1 whole cell lysate (-ve control)
Lane 2 : Raji whole cell lysate (-ve control)
Lane 3 : Jurkat whole cell lysate
Lane 4 : Human Thymus tissue lysate
Lane 5 : Mouse Thymus tissue lysate
Lane 6 : Rat Thymus tissue lysate
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 23 kDa
Additional bands at: 17 kDa. We are unsure as to the identity of these extra bands.Lanes 1 - 6: Merged signal (red and green). Green – ab16044 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab16044 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.
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All lanes : Anti-CD3 antibody (ab16044) at 1 µg/ml
Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg
Lane 2 : Thymus (Mouse) Tissue Lysate at 20 µg
Lane 3 : Thymus (Rat) Tissue Lysate at 20 µg
Secondary
All lanes : Anti-Rabbit IgG VHH Single Domain (HRP) (ab191866) at 1/50000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 23 kDa
Additional bands at: 18 kDa, 48 kDa, 62 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 8 minutesThis blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab16044 overnight at 4°C. Antibody binding was detected using an anti-rabbit IgG VHH single domain antibody conjugated to HRP, and visualised using ECL development solution ab133406.
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CD3 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to CD3 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab16044.
Secondary: Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.
Band: 23kDa; CD3