PA5-32264

antibody from Invitrogen Antibodies

Targeting: ANXA1 ANX1, LPC1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-32264

Provider

Invitrogen Antibodies

Product name

Annexin A1 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 30 min at room temperature. A suggested positive control is placenta tissue.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

500 µL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Annexin A1 Polyclonal Antibody(Product # PA1-37083) and a 38kDa band corresponding to Annexin A1 was observed across cell lines tested. Membrane enriched extracts (30ug µg lysate) of K-562 (Lane 1), A-431 (Lane 2), U-2 OS (Lane 3), IMR-32 (Lane 4), SH-SY5Y (Lane 5), HeLa (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 Dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).Expression of ANXA1 was observed all cell lines except IMR-32 and SH-5Y5Y which have no expression of ANXA1.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

CRISPR-Cas9 mediated genome editing ofANXA1 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR799825_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of ANXA1 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with ANXA1 Lentiviral sgRNA (Lane 3) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Annexin A1 Polyclonal Antibody (Product # PA5-32264) using 1:1000 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:5000 dilution).Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). A loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toANXA1 (Fig (b)).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Annexin A1 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Annexin A1 Polyclonal Antibody (Product # PA1-37083) at 5 µg in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000), for 45 minutes at room temperature (Panel a: Blue). Nuclei (Panel b: Green) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nucleus localization. Panel e represents SH-SY5Y with no expression of ANXA1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of Annexin I using anti-Annexin I Polyclonal Antibody (Product # PA5-32264) in Placenta Tissue. The recommened dilution for this antibody in immunohistochemistry applications is 1:25.