Anti-Ubiquitin Polyclonal Antibody is a Rabbit antibody targeting Ubiquitin. Anti-Ubiquitin Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM.

IgG

Human,Mouse,Rat (predicted:Pig)

1. Blank control (black line): HepG2. Primary Antibody (green line): Rabbit Anti-Ubiquitin antibody (TMAB-01928)
Dilution: 1 μg/Test;
Secondary Antibody: Goat anti-rabbit IgG-AF488
Dilution: 0.5 μg/Test.
Negative control (white blue line): PBS
Isotype control (orange line): Normal Rabbit IgG
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature.
2. Blank control (Black line): Molt4 (Black).
Primary Antibody (green line): Rabbit Anti-Ubiquitin antibody (TMAB-01928)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature.
3. Sample: Ubiquitin protein at 100ng;
Primary: Anti-Ubiquitin (TMAB-01928) at 1:300 dilution;
Secondary: HRP conjugated Goat-Anti-rabbit Igg (secondary antibody) at 1: 5000 dilution;
Predicted band size: 8.5 kDa
Observed band size: 8.5 kDa
4. Paraformaldehyde-fixed, paraffin embedded (rat colon tissue); 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 (Ubiquitin) Polyclonal Antibody, Unconjugated (TMAB-01928) at 1:400 overnight at 4°C, followed by a conjugated secondary for 20 min and DAB staining.
5. Paraformaldehyde-fixed, paraffin embedded (Human skin carcinoma); 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 (Ubiquitin) Polyclonal Antibody, Unconjugated (TMAB-01928) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
6. Paraformaldehyde-fixed, paraffin embedded (Mouse pancreas); 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 (Ubiquitin) Polyclonal Antibody, Unconjugated (TMAB-01928) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
7. HepG2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (Ubiquitin) polyclonal Antibody, Unconjugated (TMAB-01928) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.

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

Polyclonal

KLH conjugated synthetic peptide: human Ubiquitin

Human

UBB

Polyubiquitin-B

Proteasome,Ubiquitin

Ubiquitin exists either covalently attached to another protein, or free (unanchored). When covalently bound, it is conjugated to target proteins via an isopeptide bond either as a monomer (monoubiquitin), a polymer linked via different Lys residues of the ubiquitin (polyubiquitin chains) or a linear polymer linked via the initiator Met of the ubiquitin (linear polyubiquitin chains). Polyubiquitin chains, when attached to a target protein, have different functions depending on the Lys residue of the ubiquitin that is linked: Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in lysosomal degradation; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses as well as in signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Met lead to cell signaling. Ubiquitin is usually conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed. When polyubiquitin is free (unanchored-polyubiquitin), it also has distinct roles, such as in activation of protein kinases, and in signaling.