Anti-CD34 抗体 [EP373Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD34 antibody [EP373Y] (AB81289)

Flow cytometry overlay histogram showing left HUVEC positive cells and right negative HL60 stained with ab81289 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 mins. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab81289) (1x 10⁶ in 100μl at 0.2μg/ml (1/11550 dilution)) for 30 mins at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30 mins at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD34 antibody [EP373Y] (AB81289)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling CD34 with purified ab81289 at 1/2500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as the secondary antibody at 1/500. Negative control using PBS instead of primary antibody. Counter stained with Hematoxylin.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CD34 antibody [EP373Y] (AB81289)

Immunocytochemistry/Immunofluorescence analysis of HUVEC (Human umbilical vein endothelial cell line) cells labelling CD34 with purified ab81289 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counter stain.

Control : Secondary antibody only, ab150120, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-CD34 antibody [EP373Y] (AB81289)

Immunocytochemistry/Immunofluorescence analysis of human embryonic stem cell-derived endothelial cells labeling CD34 with unpurified ab81289 at 1/200. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% aTriton X-100. An Alexa Fluor® 647-conjugated secondary antibody was used at a 1/400 dilution. DAPI (blue) was used as the nuclear counter stain.

Image from Földes G et al. PLoS One. 2010 May 5;5(5):e10501 Fig 3.

• IP

Unknown

Immunoprecipitation - Anti-CD34 antibody [EP373Y] (AB81289)

CD34 was immunoprecipitated from TF-1 (Human bone marrow erythroleukemia cells) cells using purified ab81289 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab81289. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as secondary antibody at 1/1000 dilution. Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-CD34 antibody [EP373Y] (ab81289)

Predicted band size: 40 kDa

Observed band size: 120 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD34 antibody [EP373Y] (AB81289)

IHC image of CD34 staining in Mouse normal brain formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab81289, 1/250 dilution, for 15 mins at room temperature. A Goat anti-Rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-CD34 antibody [EP373Y] (AB81289)

Immunohistochemical analysis of 4% PFA-fixed 0.2% Triton X-100 permeabilized frozen Rat kidney tissue labeling CD34 with ab81289 at 1/50 (11.04 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on rat kidney. is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

AbReview54850****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD34 antibody [EP373Y] (AB81289)

Paraformaldehyde-fixed, paraffin-embedded mouse kidney tissue stained for CD34 using ab81289 at 1/200 dilution in immunohistochemical analysis.

This image is courtesy of an Abreview submitted by Rudolf Jung.

• IHC-P

AbReview63093****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD34 antibody [EP373Y] (AB81289)

Paraformaldehyde-fixed, paraffin-embedded mouse prostate tissue stained for CD34 using ab81289 at 1/150 dilution in immunohistochemical analysis.

This image is courtesy of an anonymous Abreview.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-CD34 antibody [EP373Y] (AB81289)

Immunohistochemical analysis of 4% PFA-fixed 0.2% Triton X-100 permeabilized frozen Rat lung tissue labeling CD34 with ab81289 at 1/50 (11.04 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on rat lung. is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD34 antibody [EP373Y] (AB81289)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling CD34 with purified ab81289 at 1/2500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as the secondary antibody at 1/500. Negative control using PBS instead of primary antibody. Counter stained with Hematoxylin.

• WB

Lab

Western blot - Anti-CD34 antibody [EP373Y] (AB81289)

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

Negative control : Daudi, HL-60.

Low expression : HUVEC, Neuro-2a.

All lanes:

Western blot - Anti-CD34 antibody [EP373Y] (ab81289) at 1/1000 dilution

Lane 1:

KG-1a (human bone marrow myeloblast) whole cell lysate at 20 µg

Lane 2:

TF-1 (human erythroleukemia erythroblast) whole cell lysate at 20 µg

Lane 3:

Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 4:

HL-60 (Human acute promyelocytic leukemia promyeloblast) whole cell lysate at 20 µg

Lane 5:

HUVEC (Human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 6:

bEnd.3 (mouse brain endothelioma) whole cell lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 8:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 80-120 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-CD34 antibody [EP373Y] (AB81289)

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

This product failed to detect any signal in the kidney lysate.

All lanes:

Western blot - Anti-CD34 antibody [EP373Y] (ab81289) at 1/1000 dilution

Lane 1:

Mouse lung tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse heart tissue lysate at 20 µg

Lane 4:

Rat lung tissue lysate at 20 µg

Lane 5:

Rat kidney tissue lysate at 20 µg

Lane 6:

Rat heart tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 80-120 kDa

false

Exposure time: 40s

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-CD34 antibody [EP373Y] (AB81289)

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.