Recombinant Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175)

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 (1:10000) -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 (1:10000) -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

Proteins (20 μg/well) were separated on 4–12% Bis-Tris Plus SDS gels and transferred to 0.45 μm polyvinylidene difluoride membranes. The same positive control (20 µg protein of 1% O2 hypoxia-treated T24 cell lysate) was run on each gel to normalize signals between the blots. Following transfer, membranes were cut into strips to identify the HIF pathway proteins simultaneously (examples of original Western blots are shown in Figure S1). Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate. Protein bands were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, USA) and the Alliance 4.7 imaging system, and quantified with ImageJ software.



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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) below.

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