Anti-PRMT1 antibody

• ICC

Lab

Immunocytochemistry - Anti-PRMT1 antibody (AB73246)

ab73246 staining PRMT1 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab73246 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PRMT1 antibody (AB73246)

IHC image of PRMT1 staining in Human Hippocampus FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab73246, 5µg/ml, 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

• WB

Lab

Western blot - Anti-PRMT1 antibody (AB73246)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 1% milk before being incubated with ab73246 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

This antibody was raised against an immunogen that is predicted to recognize isoforms 1,2,3 and 4 of human PRMT1. The predicted molecular weights of isoforms 1,2,3 and 4 are 41kDa, 39kDa, 39kDa and 40kDa respectively.

All lanes:

Western blot - Anti-PRMT1 antibody (ab73246) at 1 µg/mL

Lane 1:

Caco-2 (Human colonic carcinoma cell line) Whole Cell Lysate (ab76828) at 10 µg

Lane 2:

SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate (ab76999) at 10 µg

Lane 3:

Thymus (Mouse) Tissue Lysate (ab76823) at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 42 kDa

Observed band size: 25 kDa,38 kDa,43 kDa,90 kDa

true

Exposure time: 2min

• IP

Unknown

Immunoprecipitation - Anti-PRMT1 antibody (AB73246)

PRMT1 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to PRMT1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab73246.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 42kDa : PRMT1; non specific - 30kDa : We are unsure as to the identity of this extra band.

All lanes:

Immunoprecipitation - Anti-PRMT1 antibody (ab73246)

Predicted band size: 42 kDa

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• WB

Lab

Western blot - Anti-PRMT1 antibody (AB73246)

Western blot : Anti-PRMT1 antibody (ab73246) staining at 1 ug/ml, shown in black. In Western blot, ab73246 was shown to bind specifically to PRMT1. 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 after ECL incubation with 20 minute exposure. Secondary antibodies used were VeriBlot for IP Detection Reagent (HRP) (ab131366).

All lanes:

Western blot - Anti-PRMT1 antibody (ab73246) at 1 µg/mL

Lane 1:

HEK-293 input cell lysate at 10 µg

Lane 2:

HEK-293 pulldown with ab73246 cell lysate at 10 µL

Lane 3:

HEK-293 pulldown with <a href='/en-us/products/primary-antibodies/prmt1-antibody-epr18344-ab190892'>ab190892</a> cell lysate at 10 µL

Lane 4:

HEK-293 pulldown with <a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a> isotype control cell lysate at 10 µL

Lane 5:

A549 input cell lysate at 10 µg

Lane 6:

A549 pulldown with ab73246 cell lysate at 10 µL

Lane 7:

A549 pulldown with <a href='/en-us/products/primary-antibodies/prmt1-antibody-epr18344-ab190892'>ab190892</a> cell lysate at 10 µL

Lane 8:

A549 pulldown with <a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a> isotype control cell lysate at 10 µL

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>)

false