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19758
USP39 Antibody
Primary Antibodies

Polyclonal Antibody - USP39 Antibody - Western Blotting, UniProt ID Q53GS9, Entrez ID 10713 #19758

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  1. WB

Western blot analysis of extracts from various cell lines using USP39 Antibody.

To Purchase # 19758S

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa) 65
SOURCE Rabbit

Product Usage Information

Application Dilutions
Western Blotting 1:1000

Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Protocol

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Western Blotting Protocol

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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  15. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883)by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

Western Blot Reprobing Protocol

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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H20. Make buffer fresh just prior to use.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

revised June 2016

Protocol Id: 263

Specificity / Sensitivity

USP39 Antibody recognizes endogenous levels of total USP39 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val88 of human USP39 protein. Antibodies are purified by protein A and peptide affinity chromatography.

Background

Ubiquitin specific protease 39 (USP39) is a 65 kDa protein that plays an important role in pre-mRNA splicing, as well as mitotic spindle formation. It displays significant homology with ubiquitin C-terminal hydrolase proteins (UCHs), containing both an N-terminal zinc finger domain as well as UCH-1 and UCH-2-like domains also observed in the UCH2 family of proteins (1). However, USP39 lacks a catalytic cysteine residue found in UCHs and has been shown experimentally to lack peptidase activity (2). USP39 associates with the U4/U6-U5 tri-small nuclear ribonucleoprotein (U4/U6-U5 tri-snRNP) complex and is necessary for the formation of the mature spliceosome. Silencing of USP39 has been shown to adversely affect chromosome segregation and cytokinesis in U2OS cells, likely due to improper splicing of Aurora B and other mRNAs necessary for mitotic spindle formation and checkpoint function (2). In addition, USP39 has been found to be overexpressed in many types of cancers, and in most cases is associated with tumor progression and poor prognosis. Overexpression has been observed in pancreatic (3), prostate (4), colorectal (5,6), lung (6,7), gastric (8), and triple negative breast cancers (9), as well as melanoma (10) and hepatocellular carcinoma (11,12).

  1. Makarova, O.V. et al. (2001) EMBO J 20, 2553-63.
  2. van Leuken, R.J. et al. (2008) Cell Cycle 7, 2710-9.
  3. Cai, J. et al. (2017) Biochem Biophys Res Commun 486, 184-90.
  4. Huang, Y. et al. (2016) Oncotarget 7, 22016-30.
  5. Yuan, X. et al. (2017) Oncol Rep 37, 2398-2404.
  6. Fraile, J.M. et al. (2017) J Biol Chem 292, 4164-75.
  7. Lin, Z. et al. (2016) Mol Cell Biochem 422, 97-107.
  8. Wang, X. et al. (2016) Mol Med Rep 14, 301-6.
  9. Liu, S. et al. (2015) Oncol Rep 33, 2477-83.
  10. Zhao, Y. et al. (2016) Tumour Biol 37, 13167-76.
  11. Yuan, X. et al. (2015) Oncol Rep 34, 823-32.
  12. Pan, Z. et al. (2015) Biol Res 48, 18.

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