Anti-Bcl-2 antibody [E17]

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Immunohistochemical analysis of Human salivary glands taken from patients with primary Sjögren's syndrome, staining Bcl-2 with unpurified ab32124.
Antigen retrieval was performed via heat mediation in a citrate buffer (pH 6). Sections were blocked using 2% BSA, 10% normal serum and permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/100) for one hour at room temperature. An Alexa Fluor^® 594-conjugated anti-rabbit IgG was used as the secondary antibody.
N.B. Panels B and D are higher magnifications of panels A and C, respectively.

Image from Szyszko EA et al. Arthritis Res Ther. 2011 Jan 7;13(1):R2. Fig 5.; doi:10.1186/ar3220; 7 January 2011 Arthritis Research & Therapy 2011 13:R2.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Immunohistochemical analysis of formalin fixed paraffin embedded human lymphoma labelling Bcl-2 with ab32124 at a concentration of 1µg/ml.

The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab32124 anti- Bcl-2 antibody [E17] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Tissue Microarrays for Anti-Bcl2 antibody [E17] using ab32124 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pretreated with heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The section was incubated with ab32124 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B-cell lymphoma tissue labelling Bcl-2 with purified ab32124 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Bcl-2 with unpurified ab32124 at 1/200 dilution. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B cell lymphoma tissue labelling Bcl-2 with unpurified ab32124. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.

• IHC-P

AbReview52227****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Formaldehyde-fixed, paraffin-embedded human DLBCL U2932 cell line xenograft tissue stained for Bcl-2 using ab32124 at 1/200 dilution in immunohistochemical analysis, followed by Goat anti-Rabbit IgG Alexa Fluor^® 555.

This image is courtesy of an anonymous Abreview.

• IP

Unknown

Immunoprecipitation - Anti-Bcl-2 antibody [E17] (AB32124)

ab32124 (purified) at 1/30 immunoprecipitating Bcl-2 in Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-Bcl-2 antibody [E17] (ab32124)

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] (AB32124)

Bcl-2 expression determined by immunohistochemical analyses of the 4 human UM xenografts (between 3 to 5 tumors have been studied per condition).

Image from Némati F et al. PLoS One. 2014;9(1):e80836. Fig 2.; doi: 10.1371/journal.pone.0080836.

• WB

Lab

Western blot - Anti-Bcl-2 antibody [E17] (AB32124)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

MCF-7 cells express Bcl-2, while SK-BR-3 cells do not express Bcl-2 (PMID : 18430249)

All lanes:

Western blot - Anti-Bcl-2 antibody [E17] (ab32124) at 1/200 dilution

Lane 1:

MCF7 (human breast adenocarcinoma cell line) cell lysate at 10 µg

Lane 2:

SK-BR-3 (human mammary gland adenocarcinoma cell line) cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• WB

Lab

Western blot - Anti-Bcl-2 antibody [E17] (AB32124)

Lanes 1- 2 : Merged signal (red and green). Green - ab32124 observed at 26 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32124 was shown to react with BCL2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255364 (knockout cell lysate ab263752) was used. Wild-type HeLa and BCL2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32124 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Bcl-2 antibody [E17] (ab32124) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BCL2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human BCL2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-bcl2-knockout-hela-cell-line-ab255364'>ab255364</a>)

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• WB

Lab

Western blot - Anti-Bcl-2 antibody [E17] (AB32124)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Bcl-2 antibody [E17] (ab32124) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma cell line) cell lysate at 20 µg

Lane 2:

A431 (human epidermoid carcinoma cell line) cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• WB

Lab

Western blot - Anti-Bcl-2 antibody [E17] (AB32124)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32124 observed at 26 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32124 was shown to specifically react with BCL2 when BCL2 knockout samples were used. Wild-type and BCL2 knockout samples were subjected to SDS-PAGE. ab32124 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging. 3% milk used as blocking agent.

All lanes:

Western blot - Anti-Bcl-2 antibody [E17] (ab32124)

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

BCL2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

THP-1 whole cell lysate at 20 µg

Predicted band size: 26 kDa

false

• WB

Unknown

Western blot - Anti-Bcl-2 antibody [E17] (AB32124)

All lanes:

Western blot - Anti-Bcl-2 antibody [E17] (ab32124) at 1/1000 dilution

All lanes:

Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• WB

Lab

Western blot - Anti-Bcl-2 antibody [E17] (AB32124)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Bcl-2 antibody [E17] (ab32124) at 1/10000 dilution

Lane 1:

Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate at 20 µg

Lane 2:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Lane 3:

SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Bcl-2 antibody [E17] (AB32124)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.