Anti-CD70 抗体 [EPR26536-122] (BSA and Azide free) (ab300084)

All lanes : Anti-CD70 antibody [EPR26536-122] (ab300083) at 1/1000 dilution

Lane 1 : U266B1 (human multiple myeloma B lymphocyte) whole cell lysate
Lane 2 : SK-OV-3 (human ovarian cancer epithelial cell) whole cell lysate
Lane 3 : A-498 (Human kidney carcinoma) whole cell lysate
Lane 4 : 786-O (Human kidney renal cell adenocarcinoma) whole cell lysate
Lane 5 : Jurkat (human T cell leukemia T lymphocyte) whole cell lysate
Lane 6 : SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/5000 dilution

Predicted band size: 21 kDa
Observed band size: 27, 54, 81 kDa why is the actual band size different from the predicted?

This data was developed using ab300083, the same antibody clone in a different buffer formulation. 

5% NFDM/TBST was used as blosking and diluting buffer. 

Samples are non-boiled as boiling may cause protein aggregates.
The expression profile/molecular weight observed is consistent with that described in the literature (PMID: 25691774, 11980654).
Low expression: Jurkat, SH-SY5Y ( PMID: 16892042). 

Exposure time: 

Lane1: 48 seconds;
Lane2: 10 seconds

Anti-CD70 antibody [EPR26536-122] (ab300083) at 1/1000 dilution + Human lymphoma tissue lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/5000 dilution

Predicted band size: 21 kDa
Observed band size: 27, 54, 81 kDa why is the actual band size different from the predicted?


Exposure time: 81 seconds

This data was developed using ab300083, the same antibody clone in a different buffer formulation. 

5% NFDM/TBST was used as blocking and diluting buffer. 

This blot was developed using a higher sensitivity ECL substrate.
Samples are non-boiled as boiling may cause protein aggregates.
The expression profile/molecular weight observed is consistent with that described in the literature (PMID: 25691774, 11980654).

This data was developed using ab300083, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human NKT lymphoma tissue labeling CD70 with ab300083 at 1/500 dilution (0.868 µg/ml), followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining in human NKT lymphoma is observed. The section was incubated with ab300083 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.  Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used. 

This data was developed using ab300083, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human lymph node tissue labeling CD70 with ab300083 at 1/500 dilution (0.868 µg/ml), followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining in human lymph node is observed. The section was incubated with ab300083 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used. 

This data was developed using ab300083, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded clear cell carcinoma tissue labeling CD70 with ab300083 at 1/500 dilution (0.868 µg/ml), followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining in clear cell carcinoma of human kidney is observed. The section was incubated with ab300083 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.  Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used. 

This data was developed using ab300083, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling CD70 with ab300083 at 1/500 dilution (0.868 µg/ml), followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection). Positive staining in human lung cancer is observed. The section was incubated with ab300083 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used. 

This data was developed using ab300083, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized A-498 (human kidney epithelial) cells labeling CD70 with ab300083 at 1/50 (8.68 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal showing cytoplasmic and membranous staining in A-498 cell line. Negative control: Jurkat (PMID: 15226368). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.

This data was developed using anti CD70 antibody [EPR26536-122] the same antibody clone in a different buffer formulation.

CD70 was immunoprecipitated from 0.35 mg A-498 (Human kidney carcinoma) whole cell lysate with anti CD70 antibody ab300083 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300083 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A-498 (Human kidney carcinoma) whole cell lysate 10 µg (Inset)

Lane 2: ab300083 IP in A-498 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300083 in A-498 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 32 seconds

The CD27 monomer and dimer were enriched by IP.