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48160
DNA Polymerase θ (POLQ) (153-5-1) Mouse mAb
Primary Antibodies
Monoclonal Antibody

DNA Polymerase θ (POLQ) (153-5-1) Mouse mAb #48160

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  1. WB
  2. IP
Western blot analysis of extracts from mouse stimulated splenic B cells, wild-type (-) or POLQ knockout (+), using DNA Polymerase θ (POLQ) (153-5-1) Mouse mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). Lysates provided by Drs. Mélanie Prodhomme, Richard D. Wood, and Kevin McBride, The University of Texas MD Anderson Cancer Center.
Western blot analysis of extracts from A20, MEF, and NIH/3T3 cells using DNA Polymerase θ (POLQ) (153-5-1) Mouse mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Immunoprecipitation of POLQ protein from A20 cell extracts. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is DNA Polymerase θ (POLQ) (153-5-1) Mouse mAb. Western blot analysis was performed using DNA Polymerase θ (POLQ) (153-5-1) Mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody. Immunoprecipitation with primary antibody was performed for 2 hr at 4°C.
To Purchase # 48160
Cat. # Size Qty. Price
48160S
100 µl

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa) 280
Source/Isotype Mouse IgG1 kappa

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100

Storage

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.

Protocol

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Western Blotting Protocol For High Molecular Weight Proteins

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 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. 1X Tris-Acetate SDS Running Buffer: To prepare 1 L 1X running buffer: add 6.06 g Tris Base, 8.96 g Tricine, and 1.0 g SDS to a total volume of 1L using dH2O, mix. Adjust pH to 8.24.
  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. Color-coded Prestained Protein Marker, High Range (43-315 kDa): (#12949).
  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-mouse IgG, HRP-linked Antibody (#7076).
  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 a 3-8% Tris-Acetate, SDS-PAGE gel (8 cm x 13 cm), using 1X Tris-Acetate SDS Running Buffer.

    NOTE: Loading of prestained molecular weight markers (#12949, 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).
    1. When using a semi-dry transfer apparatus, select the high molecular weight transfer protocol if available.
    2. When using a wet transfer method, reduce the methanol in the transfer buffer to 5% (v/v) and transfer for 3 hours at 70V constant voltage, at 4°C.

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 webpage) 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-mouse IgG, HRP-linked Antibody (#7076 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

  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.

Protocol Id: 2884

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins utilizing Protein G agarose beads for subsequent analysis by western immunoblot or kinase activity.

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 of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.

    NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

  3. 3X 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.
  4. Protein G Agarose Beads: (#37478).
  5. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 μl 10X kinase buffer to 900 μl dH2O, mix.
  6. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 μM), add 10 μl ATP (10 mM) to 490 μl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

  1. Vortex to mix beads.
  2. Add 10–30 μl of 50% Protein G agarose bead slurry to 200 μl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30–60 min.
  4. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
  5. Proceed to immunoprecipitation below.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 μl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Add Protein G agarose (10–30 μl of 50% bead slurry). Incubate with rotation for 1–3 hr at 4°C.
  3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to sample analysis by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 μl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: When using primary antibodies produced in rabbit to to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse heavy or light chain.

When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 μl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 μl 1X kinase buffer supplemented with 200 μM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 μl) on SDS-PAGE (4–20%).

posted October 2016

revised October 2021

Protocol Id: 1244

Specificity / Sensitivity

DNA Polymerase θ (POLQ) (153-5-1) Mouse mAb recognizes endogenous levels of total POLQ protein. By western blot, this antibody detects a band of unknown origin at approximately 260 kDa.

Species Reactivity:

Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human POLQ protein.

Background

Mammalian cells undergo DNA damage in response to various intrinsic and extrinsic forces, and signaling pathways are in place to sense and repair damaged DNA to avoid genome instability and mutation. Repair of toxic DNA double-strand breaks (DSBs) can occur through several pathways, and pathway choice may depend on the cell cycle phase and the nature of the DSB (1). The two main pathways for DSB repair are classical non-homologous end joining (C-NHEJ) and homologous recombination (HR). In addition, three error-prone pathways, single-strand annealing (SSA), microhomology-mediated end-joining (MMEJ), and polymerase theta-mediated end-joining (TMEJ) have been described (2).

Exploiting DNA repair deficiencies in human cancer by targeting intact DNA repair pathways is a promising approach to cancer therapy. This approach has been successful with the use of poly (ADP-ribose) polymerase (PARP) inhibitors in HR-deficient cancer (3).DNA polymerase θ, encoded by the POLQ gene, is a 290 kDa protein with an N-terminal helicase-like domain and a C-terminal DNA polymerase domain (4). ZEB1, an epithelial-mesenchymal transition (EMT) transcription factor, has been shown to regulate TMEJ by suppressing POLQ expression (5). DNA polymerase θ is essential in TMEJ and is, therefore, an important target for drug development in HR-deficient cancers (6,7). Several inhibitors of DNA polymerase θ are under investigation in pre-clinical and clinical trials (8,9).

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