Anti-CD33 antibody [EPR4423]

• Flow Cyt

Lab

Flow Cytometry - Anti-CD33 antibody [EPR4423] (AB134115)

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte, Left) / THP1 (Human monocytic leukemia monocyte, Right) labeling CD33 with ab134115 at 1/500 dilution (red). Goat anti rabbit IgG (Alexa Fluor^® 647, ab150079) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (ab172730) was used as isotype control (Black). Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue).

Negative control : Jurkat.
Gated on viable cells.

• IP

Unknown

Immunoprecipitation - Anti-CD33 antibody [EPR4423] (AB134115)

ab134115 (purified) at 1/60 immunoprecipitating CD33 in 10 μg THP-1 (Lanes 1 and 2, observed at 67-75 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. Blocking buffer and concentration : 5% NFDM/TBST Dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-CD33 antibody [EPR4423] (ab134115)

Predicted band size: 40 kDa

false

• WB

Lab

Western blot - Anti-CD33 antibody [EPR4423] (AB134115)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-CD33 antibody [EPR4423] (ab134115) at 1/5000 dilution

Lane 1:

THP-1 cell lysate at 10 µg

Lane 2:

U937 cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 40 kDa

Observed band size: 67 kDa,75 kDa

false

• WB

Supplier Data

Western blot - Anti-CD33 antibody [EPR4423] (AB134115)

False colour image of Western blot : Anti-CD33 antibody [EPR4423] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab134115 was shown to bind specifically to CD33. A band was observed at 60-80 kDa in wild-type THP-1 cell lysates with no signal observed at this size in CD33 knockout cell line ab273831 (knockout cell lysate ab273785). To generate this image, wild-type and CD33 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CD33 antibody [EPR4423] (ab134115) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 10 µg

Lane 2:

Western blot - Human CD33 knockout THP-1 cell lysate (ab273785) at 10 µg

Lane 3:

HL-60 cell lysate at 10 µg

Lane 4:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 40 kDa

Observed band size: 60-80 kDa

false

• WB

Unknown

Western blot - Anti-CD33 antibody [EPR4423] (AB134115)

All lanes:

Western blot - Anti-CD33 antibody [EPR4423] (ab134115) at 1/1000 dilution

Lane 1:

THP1 cell lysate at 10 µg

Lane 2:

U937 cell lysate at 10 µg

Lane 3:

HL60 cell lysate at 10 µg

Lane 4:

HepG2 cell lysate at 10 µg

Lane 5:

HeLa cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 40 kDa

Observed band size: 67-75 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-CD33 antibody [EPR4423] (AB134115)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.