Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Apolipoprotein L 1/APOL1 with ab252218 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on glomerulus and weak cytoplasmic staining on renal tubules (PMID: 21997392, PMID: 28724794, PMID: 27138898) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Apolipoprotein L 1/APOL1 with ab252218 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining mainly in plasma of human colon blood vessels is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

IHC image of Apolipoprotein L1/APOL1 staining in a section of frozen normal human kidney performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab252218, 1/1000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Apolipoprotein L 1/APOL1 was immunoprecipitated from 0.35 mg of HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate with ab252218 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab252218 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HepG2 whole cell lysate 10 μg (Input).
Lane 2: ab252218 IP in HepG2 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252218 in HepG2 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

All lanes : Anti-Apolipoprotein L1/APOL1 antibody [EPRBOB-2-F1-17] (ab252218) at 1/1000 dilution

Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 2 : HDLM-2 (human Hodgkin lymphoma cell line) whole cell lysate
Lane 3 : Human liver lysate
Lane 4 : Human plasma

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 44 kDa
Observed band size: 43 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 15 seconds.

Lanes 2 & 3: 136 seconds.

Lane 4: 3.25 seconds.

The expression profile/ molecular weight observed is consistent with what has been described in the literature and the doublet band lane 4 might be the different isoform of ApoL1 (PMID: 25012173, 23300552).