Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD69 with ab233396 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining in lymphoid cells of human tonsil (PMID: 2807372) is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-CD69 antibody [EPR21814] (ab233396) at 1/5000 dilution

Lane 1 : Un-treated Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate
Lane 2 : Daudi treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 23 kDa
Observed band size: 28,32 kDa why is the actual band size different from the predicted?


Exposure time: 92 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

PMA treatment increases the basal level of p28/32 on Daudi. PMID: 1617156.

All lanes : Anti-CD69 antibody [EPR21814] (ab233396) at 1/1000 dilution

Lane 1 : HL-60 (human Acute Promyelocytic Leukemia promyeloblast) whole cell lysate at 20 µg
Lane 2 : K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg
Lane 3 : U937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg
Lane 4 : H9 (human lymphoma T lymphocyte) whole cell lysate at 10 µg
Lane 5 : Human tonsil at 20 µg
Lane 6 : Human lymphoma at 20 µg
Lane 7 : Human thymus at 20 µg
Lane 8 : Human colon at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 23 kDa
Observed band size: 24, 32 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-5 and 7-8: 3 minutes; Lane 6: 26 seconds.

We observe an extra band at approximately 15 kDa that we do not yet have an explanation for; it could be non-specific.

Immunohistochemical analysis of paraffin-embedded human cervix cancer tissue labeling CD69 with ab233396 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining in infiltrated lymphoid cells of human cervical cancer is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

CD69 was immunoprecipitated from 0.35 mg Daudi (human Burkitt's lymphoma lymphoblast) treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate with ab233396 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab233396 at 1/5,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution.

Lane 1: Daudi (human Burkitt's lymphoma lymphoblast) treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate 10 µg (Input).
Lane 2: ab233396 IP in Daudi treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab233396 in Daudi treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, ab120297) for 24 hours whole cell lysate (-).

Blocking/Dilution buffer: 5% NFDM/TBST.