Recombinant Anti-p27 KIP 1 antibody [EPFHCR16] (ab92741)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:500) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:500) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Cells were lysed in the protein lysis buffer (Xinyu Biotechnology, Shanghai, China). The protein concentration was determined using the BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA). Protein was separated with 10% SDS-PAGE, transferred to a PVDF membrane (Millipore), and then blocked in 5% nonfat dried milk in TBST for 2 h. The PVDF membrane was then incubated with primary antibodies (Rabbit monoclonal anti-CyclinD1, cat no. ab137875, dilution: 1:1000; Rabbit monoclonal anti-P21, cat no. ab109520, dilution: 1:500; Rabbit monoclonal anti-P27, cat no. ab92741, dilution: 1:500; Rabbit polyclonal anti-P53, cat no. ab31333, dilution: 1:200; and mouse monoclonal anti-GAPDH, cat no. ab8245, dilution: 1:3000. All the antibodies were from Abcam (Cambridge, UK)) at 4°C overnight, and then washed with TBST four times. Then, the PVDF membrane was incubated with mouse anti-rabbit secondary antibody for 1 h at room temperature, and then washed with TBST four times. All antibodies were purchased from Thermo Fisher, USA. The immune complexes were then detected using the ECL Western Blotting Kit (Pierce) and X-film (Kodak, Tokyo, Japan). ImageJ software (NIH) was used to analyze the relative protein expression, represented as the density ratio versus GAPDH.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Western blotting CDKN1B using Recombinant Anti-p27 KIP 1 antibody [EPFHCR16] (ab92741) from Abcam.

Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-p27 KIP 1 antibody [EPFHCR16] (ab92741) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Western blotting CDKN1B using Recombinant Anti-p27 KIP 1 antibody [EPFHCR16] (ab92741) from Abcam. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.