Antibody Type: Rabbit Polyclonal

Application: WB,IHC-P,IHC-Fr,IF,FCM

Reactivity: Human,Mouse,Rat (predicted:Chicken,Dog,Pig,Cow,Horse,Rabbit,Sheep,Goat)

IgG

Human,Mouse,Rat (predicted:Chicken,Dog,Pig,Cow,Horse,Rabbit,Sheep,Goat)

1. Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ERK1 + ERK2) Polyclonal Antibody, Unconjugated (TMAB-00633) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
2. Sample:
Brain (Rat) Lysate at 30 μg
Heart (Rat) lysate at 30 μg
Primary: Anti-ERK2/MAPK1 (TMAB-00633) at 1/200 dilution
Secondary: HRP conjugated Goat-Anti-rabbit IgG (secondary antibody) at 1/3000 dilution
Predicted band size: 42 kDa
Observed band size: 42 kDa
3. Tissue/cell: human lung carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37°C for 20 min;
Incubation: Anti-ERK2/MAPK1 Polyclonal Antibody, Unconjugated (TMAB-00633) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
4. Blank control: Hep G2 cells (blue). Primary Antibody: Rabbit Anti-ERK1 + ERK2 antibody (TMAB-00633), Dilution: 1 μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit Igg (orange),used under the same conditions); Secondary Antibody: Goat anti-rabbit IgG-Pe (white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min), then permeabilized with 90% ice-cold methanol for 30 min on ice. Primary antibody (TMAB-00633, 1 μg/1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 10% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice.
5. Sample:
Lane 1: A431 (Human) Cell Lysate at 30 μg
Lane 2: MCF-7 (Human) Cell Lysate at 30 μg
Lane 3: Huvec (Human) Cell Lysate at 30 μg
Primary:
Anti-ERK1 + ERK2 (TMAB-00633) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 44/42 kDa
Observed band size: 42/40 kDa
6. Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 μg
Lane 2: Cerebrum (Rat) Lysate at 40 μg
Lane 3: LympHnode (Mouse) Lysate at 40 μg
Lane 4: LympHnode (Rat) Lysate at 40 μg
Primary:
Anti-ERK1 + ERK2 (TMAB-00633) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 44/42 kDa
Observed band size: 40 kDa

WB,IHC-P,IHC-Fr,IF,FCM

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; IF: 1:100-500; FCM: 1μg/Test

Polyclonal

KLH conjugated synthetic peptide: human ERK2

Human

5595

Tangles & Tau,MAPK Pathway,Cytoplasmic