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.

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 minutes

This 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.

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 70^oC; 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