PA5-34584

antibody from Invitrogen Antibodies

Targeting: FMR1 FMRP, FRAXA, MGC87458, POF, POF1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Recommended by provider)

Immunohistochemistry (Recommended by provider)

Antibody Data

Product number

PA5-34584

Provider

Invitrogen Antibodies

Product name

FMRP Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

For Western Blot, this antibody has non-specific bands at 13 kDa,32 kDa and 68 kDa. Heat mediated antigen retrieval recommended prior to tissue staining. This antibody is predicted to react with orangutan based on sequence homology.

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

1 mg/mL

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

Knockdown of FMRP was achieved by transfecting HEK-293 cells with FMRP specific siRNAs (Silencer® select Product # s5316, s5317). Western blot analysis (Fig. a) was performed whole cell extracts from the HEK-293 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with FMRP Polyclonal Antibody (Product # PA5-34584, 0.1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to FMRP.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-FMRP Rabbit Polyclonal Antibody (Product # PA5-34584) and a 71 kDa band corresponding to FMRP was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), U-87 MG (Lane 2), HeLa (Lane 3), A549 (Lane 4), SH-SY5Y (Lane 5), Hep G2 (Lane 6), Mouse Brain (Lane 7) and Rat Brain (Lane 8) were electrophoresed using NuPAGE™ 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 the primary antibody (0.75 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of FMRP was performed using Hep G2 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 1 hour at room temperature. The cells were labeled with FMRP Rabbit Polyclonal Antibody (Product # PA5-34584) at 5 µg/mL dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the composite image showing nuclear and cytoplasmic localization of FMRP. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.