Western blot analysis of extracts from various cell lines using PML (E5R8T) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). In agreement with RNA-seq studies, AN3-CA cells express very low levels of PML protein.
Western blot analysis of MDA-MB-231 cells expressing inducible HA-tagged PML protein (MDA-MB-231 HA-PML), either uninduced (-) or induced with doxycycline (50 ng/ml, 6 d; +), using PML (E5R8T) Rabbit mAb (upper), HA-Tag (C29F4) Rabbit mAb #3724 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, doxycycline induction increased the expression level of PML protein. Cells were kindly provided by Dr. Arkaitz Carrecedo, Natalia Martin, Laura Bozal and Amaia Ercilla.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with Myc/DDK-tagged cDNA expression constructs encoding full-length human PML protein (hPML-Myc/DDK) or full-length mouse PML protein (mPML-Myc/DDK), using PML (E5R8T) Rabbit mAb (upper) or DYKDDDDK Tag (D6W5B) Rabbit mAb #14793 (lower). As shown, PML (E5R8T) Rabbit mAb detects overexpressed human PML protein (hPML-Myc/DDK), but not mouse PML protein (mPML-Myc/DDK).
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
PML (E5R8T) Rabbit mAb recognizes endogenous levels of total PML protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro364 of human PML protein.
Promyelocytic leukemia protein (PML) functions via its association with PML bodies in the nucleus (1). PML bodies are dynamic protein aggregates that are interspersed between chromatin in the nuclei of most mammalian cells. The PML protein acts as a scaffold in PML bodies to recruit other proteins, a process regulated by post-translational modifications, such as sumoylation. PML bodies function to regulate a large number of cellular processes, such as tumor suppression, transcriptional regulation, apoptosis, senescence, DNA damage response, and viral defense (1). The chromosomal translocation t(15;17)(q21;q21) involving the fusion of PML and RARalpha is found in acute promyelocytic leukemia (APL), while a second translocation t(9;15)(p13;q24) involving the fusion of PML and PAX5 was found in acute lymphoblastic leukemia (ALL) (2-6). In addition, PML is frequently inactivated or downregulated in cancer.
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