Recombinant Anti-Musashi 1 / MSI1 antibody [EPR26106-92] (ab305170)

Blocking / Diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lysates at 20 µg per lane.
Performed under reducing conditions.

False colour image of Western blot: Anti-Musashi 1 / Msi1 antibody [EPR26106-92] (ab305170) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab305170 was shown to bind specifically to Musashi 1 / Msi1. A band was observed at 39 kDa in wild-type HAP1 cell lysates with no signal observed at this size in Musashi 1 / Msi1 knockout cell line (knockout cell lysate). To generate this image, wild-type and Musashi 1 / Msi1 knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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.

Blocking / Diluting buffer and concentration: 5% NFDM/TBST

Low expression tissues: kidney, heart (PMID: 8660864).

Blocking / Diluting buffer and concentration: 5% NFDM/TBST

Negative control: NIH/3T3 (PMID:11359897).

Blocking / Diluting buffer and concentration: 5% NFDM/TBST

Both recombinant proteins were made in-house and expressed from the E. coli expression systems.

This antibody does not cross-react with mouse Musashi 2 / Msi2.

Musashi 1 / Msi1 was immunoprecipitated from 0.35 mg mouse P0 brainstem tissue lysate 10 µg with ab305170 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305170 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: mouse P0 brainstem tissue lysate 10 µg

Lane 2: ab305170 IP in mouse P0 brainstem tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab305170 in mouse P0 brainstem tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 84 seconds.

Observed MW(KDa): 39 kDa

Flow cytometric analysis of 4% paraformaldehyde, fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast, Left) / Neuro-2a (mouse neuroblastoma neuroblast, Right) cells labeling Musashi 1 / Msi1 with AB305170 at 1/50 dilution (1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control: NIH/3T3 (PMID: 11359897).

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Musashi 1 / Msi1 with ab305170 at 1/500 (0.918 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) was used. Negative control: No staining on mouse kidney (PMID: 25717188). The section was incubated with ab305170 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Musashi 1 / Msi1 with ab305170 at 1/500 (0.918 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) was used. Positive staining on mouse testis (PMID:11804968). The section was incubated with ab305170 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded rat small intestine tissue labeling Musashi 1 / Msi1 with ab305170 at 1/500 (0.918 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) was used. Positive staining on intestinal crypts of rat (PMID:12558601). The section was incubated with ab305170 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse small intestin tissue labeling Musashi 1 / Msi1 with ab305170 at 1/500 (0.918 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) was used. Positive staining on intestinal crypts of mouse (PMID:12558601). The section was incubated with ab305170 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins