PA1-087

antibody from Invitrogen Antibodies

Targeting: HTT HD, IT15

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Recommended by provider)

Antibody Data

Product number

PA1-087

Provider

Invitrogen Antibodies

Product name

Phospho-Huntingtin (Ser421) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

PA1-087 has been successfully used in immunofluorescence, immunohistochemistry, and Western blotting applications on human and mouse samples.

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

0.24 mg/mL

Storage

-20°C

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Phospho-Htt (pSer421) was performed on 293T cell lysates from cells transfected with full-length Htt (Lanes 1 and 2) or an Htt Ser421 mutant (Lanes 3 and 4). Following transfection, cells were left untreated (Lanes 1 and 3) or treated with PPI inhibitor (Lanes 2 and 4) to increase phosphorylation of Htt at Ser421. Lysates were loaded onto a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Phospho-Htt (pSer421) polyclonal antibody (Product # PA1-087) at a dilution of 1:500 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20 and probed with a goat anti-rabbit IgG-HRP secondary antibody (Product # 31460) at a dilution of 1:30,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34087).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-Huntingtin/Htt pSer421 was performed using 70% confluent log phase SH-SY5Y cells which were serum starved (48 h) and stimulated with 50 ng/mL IGF-1 for 30 min. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-Huntingtin/Htt pSer421 Rabbit Polyclonal Antibody (Product # PA1-087) at 1:250 dilution in0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) 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 cytoplasmic localization. Panel e shows the untreated cells. Panel f shows the control without primary antibody. The images were captured at 60X magnification.