710877

antibody from Invitrogen Antibodies

Targeting: RPS6 S6

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710877

Provider

Invitrogen Antibodies

Product name

Phospho-S6 (Ser240, Ser244) Recombinant Polyclonal Antibody (11 HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Antibody clone number

11 HCLC

Vial size

100 µg

Concentration

0.5 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

Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEK-293 (1), HEK-293 (Serum starved24 hours and released for 1 hour) (2), PC3 (3), PC3 (Serum starved24 hours and released for 1 hour) (4), Caco2 (5), Caco2 (Serum starved24 hours and released for 1 hour) (6), K562 (7), K562 (Serum starved24 hours and released for 1 hour) (8), Colo-205 (9) and Colo-205 (Serum starved24 hours and released for 1 hour) (10). The blots were probed with Anti-RPS6 (pSpS240/244) Recombinant Rabbit Polyclonal Antibody (Product # 710877, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 30 kDa band corresponding to RPS6 (pSpS240/244) was observed across cell lines tested. Treatment response is more evident in cell lines where basal expression is less. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on fixed and permeabilized A375 cells which wetre serum starvation (16 hours) and serum released (1 hour) for detection of RPS6 (pSpS240/244) using Anti- RPS6 (pSpS240/244) Recombinant Rabbit Polyclonal Antibody (Product # 710877, 1-2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of RPS6 (pSpS240/244) protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of RPS6 (pSpS240/244) Panel e)shows no signal which demonstrates antibody specificity against RPS6 (pSpS240/244) phosphorylated peptide (Antibody was incubated with phosphorylated peptide for 1 hour/37C). Panel f) represents control cells with no primary Antibody to assess background.