Western blot analysis of extracts from HT-29 and U-2 OS cells, untreated (-) or synchronized by double thymidine block followed by an eight hour release into complete medium (+), using Cyclin F (D9K2U) Rabbit mAb (upper) or α-Actinin (D6F6) Rabbit mab #6487 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with Myc/DDK-tagged full-length human Cyclin F (hCyclin F-Myc/DDK; +), using Cyclin F (D9K2U) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Cyclin F (D9K2U) Rabbit mAb recognizes endogenous levels of total Cyclin F protein. This antibody recognizes an unidentified protein of 80 kDa by western blot.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro665 of human Cyclin F protein.
Cyclin F is the founding member of the F-box protein family, present in all eukaryotic cells. F-box proteins are components of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. The substrate specificity of the SCF complex is determined by the interchangeable F-box proteins, which act as adaptors by associating with phosphorylated substrate proteins and recruiting them to the SCF core (1).
Cyclin F contains a cyclin box domain in addition to an F-box domain, but does not regulate the activity of cyclin dependent kinases. Cyclin F expression does oscillate during the cell cycle, however, peaking in G2 phase (2).
Cyclin F interacts with the centrosomal protein CP110, which plays critical roles centriole duplication and spindle formation. Cyclin F-mediated degradation of CP110 in G2 phase is required for normal progression into mitosis (3). In response to ionizing radiation, which causes DNA double strand breaks, Cyclin F interacts with B-Myb, preventing cyclin A-dependent phosphorylation of B-Myb, and delaying progression into mitosis. This G2 phase arrest allows the cell to respond to the DNA damage-induced G2/M phase checkpoint (4). Cyclin F also controls the stability of the ribonucleotide reductase M2 subunit, RRM2, which functions in maintaining the levels of dNTPs available in the cell for DNA synthesis and repair, in response to genotoxic stress (5). Researchers have implicated cyclin F as a prognostic marker in hepatocellular carcinoma (HCC) (6).
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