Anti-CD3 antibody

• IP

Unknown

Immunoprecipitation - Anti-CD3 antibody (AB16044)

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

All lanes:

Immunoprecipitation - Anti-CD3 antibody (ab16044)

Predicted band size: 19 kDa

false

• WB

Lab

Western blot - Anti-CD3 antibody (AB16044)

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 VHH Single Domain Anti-Rabbit IgG Fc (HRP), and visualised using ECL development solution ab133406.

All lanes:

Western blot - 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:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (<a href='/en-us/products/secondary-antibodies/vhh-single-domain-rabbit-igg-fc-hrp-ab191866'>ab191866</a>) at 1/50000 dilution

Predicted band size: 19 kDa

Observed band size: 18 kDa,23 kDa,48 kDa,62 kDa

true

Exposure time: 8min

• WB

Lab

Western blot - Anti-CD3 antibody (AB16044)

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:

Western blot - Anti-CD3 antibody (ab16044) at 1 µg/mL

Lane 1:

THP1 whole cell lysate (-ve control) at 15 µg

Lane 2:

Raji whole cell lysate (-ve control) at 15 µg

Lane 3:

Jurkat whole cell lysate at 15 µg

Lane 4:

Human Thymus tissue lysate at 15 µg

Lane 5:

Mouse Thymus tissue lysate at 15 µg

Lane 6:

Rat Thymus tissue lysate at 15 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: 19 kDa

Observed band size: 17 kDa,23 kDa,68 kDa

false