At BioLegend, we offer antibody clones raised against phosphorylated targets that have been validated for use in multiple experimental applications. For example, clone 4B11B69 raised against phosphorylated ERK1/2 (Tyr202/Tyr204) has been validated in-house for successful use in Western blot, Immunofluorescence, and Intracellular Flow Cytometry.

Alexa Fluor® 647 anti-ERK1/2 Phospho (Thr202/Tyr204) Antibody (clone 4B11B69)

Intracellular Flow Cytometry (ICFC):

 

Human peripheral blood lymphocytes were stimulated with (filled histogram), or without (open histogram), Cell Activation Cocktail (without Brefeldin A) for 15 minutes, then fixed with Fixation Buffer, permeabilized with True-Phos™ Perm Buffer, and intracellularly stained with ERK1/2 Phospho (Thr202/Tyr204) (Clone 4B11B69) Alexa Fluor® 647.

 

C57BL/6 mouse splenocytes were stimulated with (filled histogram) or without (open histogram) Cell Activation Cocktail (without Brefeldin A) for 15 minutes, then fixed with Fixation Buffer, permeabilized with True-Phos™ Perm Buffer, and intracellularly stained with ERK1/2 Phospho (Thr202/Tyr204) (Clone 4B11B69) Alexa Fluor® 647.

Superior Signal-to-Noise Ratio:

 

Human peripheral blood lymphocytes were stimulated with (filled histogram) or without (open histogram) Cell Activation Cocktail (without Brefeldin A) for 15 minutes, then fixed with Fixation Buffer, permeabilized with True-Phos™ Perm Buffer, and intracellularly stained with Alexa Fluor® 647 ERK1/2 Phospho (Thr202/Tyr204). Clone 4B11B69 was used at 0.125 µg/test and compared side-by-side against the competitor's product, which was used at 0.1 µg/test.

 

Products Referenced Above:

Product Name Catalog #
Alexa Fluor® 647 anti-ERK1/2 Phospho (Thr202/Tyr204) Antibody 675503 | 675504
Cell Activation Cocktail (without Brefeldin A) 423301 | 423302
Fixation Buffer 420801
True-Phos™ Perm Buffer 425401

 


Purified anti-ERK1/2 Phospho (Thr202/Tyr204) Antibody (clone 4B11B69)

Western Blot (WB):

 

Left: Untreated (lane 1) and PMA-treated (lane 2) Jurkat whole cell lysates were resolved by electrophoresis, transferred to nitrocellulose, and probed with purified monoclonal anti-ERK1/2 Phospho (Thr202/Tyr204) (clone 4B11B69) antibody. Proteins were visualized using an anti-mouse-IgG secondary conjugated to HRP (Cat. No. 405306) and chemiluminescence detection.

Immunofluorescence (IF):

 

Above: Untreated (left) and PMA-treated (20 nM PMA treatment for 10 minutes, right) mouse NIH-3T3 cells were stained with purified anti-ERK1/2 Phospho (Thr202/Tyr204) (clone 4B11B69) antibody, followed by staining with DyLight™ 488 conjugated goat anti-mouse IgG (green, Cat. No. 405310) antibody and Alexa Fluor® 594 conjugated phalloidin (red). Nuclei were stained with DAPI (blue).

Products Referenced Above:

Product Name Catalog #
Purified anti-ERK1/2 Phospho (Thr202/Tyr204) Antibody 675502
HRP Goat anti-mouse IgG (minimal x-reactivity) Antibody (WB) 405306
DyLight™ 488 Goat anti-mouse IgG (minimal x-reactivity) Antibody (IF) 405310
DAPI (4', 6-Diamidino-2-Phenylindole, Dilactate) (IF) 422801

 


Phospho-target Antibodies for Neuroscience

With the acquisition of Covance's antibody product portfolio, BioLegend now offers antibodies targeted towards phosphorylated proteins implicated in the biology of the brain, and neurodegenerative diseases.

Purified anti-Tau Phospho (Thr181) Antibody Clone M7004D06

Immunohistochemistry (IHC):

 

(Left) IHC staining of anti-Tau Phospho (Thr181) antibody (clone M7004D06) on formalin-fixed paraffin-embedded Alzheimer's brain tissue. Following antigen retrieval using Sodium Citrate, the tissue was incubated with the primary antibody at 10 µg/ml for 60 minutes at room temperature. BioLegend's Ultra Streptavidin (USA) HRP Detection Kit was used for detection.

Western Blot (WB):

 

(Left) Western blot of anti-Tau Phospho (Thr181) (clone M7004D06), anti-Tau Phospho (Ser396) (clone PHF-13) antibodies, and isotype-matched control IgG1 and IgG2b. Lane 1: 20 µg of Parkinson’s disease human brain lysate; Lane 2: 2 µg of Tau paired helical filaments. Blots were incubated overnight at 4°C with 10 µg/ml of the primary antibody, followed by horseradish peroxidase labeled goat anti-mouse secondary antibody. Western blot of anti-Tau Phospho (Ser396) (clone PHF-13) serves as a positive control for Tau staining.

Products Referenced Above:

Product Name Catalog #
Purified anti-Tau Phospho (Thr181) Antibody (Clone M7004D06) 846602
Purified anti-Tau Phospho (Ser396) Antibody (Clone PHF-13) 829001
Ultra Streptavidin (USA) HRP Detection Kit (Multi-Species) (IHC) 929701
Purified anti-Tau Phospho (Ser396) Antibody  829001