Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human POLH protein (hPOLH-Myc/DDK; +), using DNA Polymerase η (E1I7T) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).Learn more about how we get our images.
Western blot analysis of extracts from various cell lines, including POLH-negative GM03617 cells, using DNA Polymerase η (E1I7T) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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 June 2016
Protocol Id: 19
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.
DNA Polymerase η (E1I7T) Rabbit mAb recognizes endogenous levels of total POLH protein. The antibody recognizes a 40 kDa background band of unknown origin. In some cell lines, the antibody recognizes a 60 kDa band of unknown origin. This band does not correspond to the expected size of truncated POLH in GM03617 cells.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro309 of human POLH protein.
A total of fifteen mammalian DNA polymerase enzymes catalyze the synthesis of nascent DNA during DNA replication and repair (1). DNA polymerase eta (POL η, POLH, Rad30) is one of a specialized type of DNA polymerases that function in DNA repair and translesion synthesis (TLS). POLH can accommodate and read through bulky DNA lesions such as pyrimidine dimers, which allows for continued DNA synthesis past lesions and limited stalling of replication forks (2,3). Damage inducing conditions, such as exposure to UV light or cisplatin, recruit POLH to sites of bulky DNA lesions where the polymerase interacts with PCNA (4,5). Mutations in the human POLH gene can result in a form of xeroderma pigmentosum (XPV), an autosomal recessive disorder characterized by hypersensitivity to light and susceptibility to skin cancer (6).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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|13848S||100 µl (10 western blots)||$ 255.0|