Membranes were blocked in 3 % milk in TBS-0.1 % Tween ® 20 (TBS-T) before incubation with primary antibodies overnight at 4 Â☌. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. To generate this image, wild-type and BRPF1 knockout HEK-293T cell lysates were analysed. A band was observed at 160, 145 and 138 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in BRPF1 knockout cell line ab266549 (knockout cell lysate ab258795). In Western blot, ab226909 was shown to bind specifically to Peregrin/BRPF1. Lane 2 : BRPF1 knockout HEK-293T cell lysateįalse colour image of Western blot: Anti-Peregrin/BRPF1 antibody - N-terminal staining at 1/500 dilution, shown in green Mouse anti-Alpha Tubulin ( ab7291) loading control staining at 1/20000 dilution, shown in red. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye ® 800CW) preabsorbed ( ab216773) and Goat anti-Mouse IgG H&L (IRDye ® 680RD) preabsorbed ( ab216776) at 1/20000 dilution.Īll lanes : Anti-Peregrin/BRPF1 antibody - N-terminal ( ab226909) at 1/500 dilution Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Membranes were blocked in 5 % milk in TBS-0.1 % Tween ® 20 (TBS-T) before incubation with primary antibodies overnight at 4 ☌. A band was observed at 120 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in BRPF1 knockout cell line ab266549 (knockout cell lysate ab258795). In Western blot, ab259840 was shown to bind specifically to Peregrin/BRPF1. Observed band size: 120 kDa why is the actual band size different from the predicted?įalse colour image of Western blot: Anti-Peregrin/BRPF1 antibody staining at 1/500 dilution, shown in green Mouse anti-Alpha Tubulin ( ab7291) loading control staining at 1/20000 dilution, shown in red. Lanes 2-3 : BRPF1 knockout HEK-293T cell lysate at 20 µg Lane 1 : Wild-type HEK-293T cell lysate at 20 µg Lane 4 : Anti-Peregrin/BRPF1 antibody ( ab259840) For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages. This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. Cells should be passaged when they have achieved 80-90% confluence. A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.A guide seeding density of 2x10 4 cells/cm 2 is recommended.All seeding densities should be based on cell counts gained by established methods.Incubate the culture at 37☌ incubator with 5% CO 2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.Ĥ. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x10 4 cells/cm 2. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. 20 mL represents maximum recommended dilution.ģ. 10 mL represents minimum recommended dilution. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. Thaw the vial in 37☌ water bath for approximately 1-2 minutes.Ģ. Storage at -80☌ may result in loss of viability.ġ. Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80☌. Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.Ĭryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.Ĭulture medium: DMEM (High Glucose) + 10% FBS Recommended control: Human wild-type HEK293T cell line ( ab255449).
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