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28630
Phospho-Syntaxin 17 (Ser202) Antibody
Primary Antibodies

Phospho-Syntaxin 17 (Ser202) Antibody #28630

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  1. WB

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expresssing Myc/DDK-tagged human syntaxin 17 (hSTX17-Myc/DDK; +) and Myc/DDK-tagged human TBK1 (hTBK1-Myc/DDK; +), using Phospho-Syntaxin 17 (Ser202) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 28630S
製品番号 サイズ 価格 在庫
28630S
100 µl

Supporting Data

REACTIVITY H
SENSITIVITY Transfected Only
MW (kDa) 40-42
SOURCE Rabbit

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • 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
  • All-All Species Expected

Product Usage Information

Application Dilution
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

Phospho-Syntaxin 17 (Ser202) Antibody recognizes transfected levels of syntaxin 17 protein only when phosphorylated at Ser202.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology:

Mouse, Rat

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser202 of human syntaxin 17 protein. Antibodies were purified by peptide affinity chromatography.

Background

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3). 

Syntaxin 17/STX17 is a SNARE factor recruited to autophagosomes and required for autophagosome fusion to lysosomes. Syntaxin 17 interacts with SNAP29 (Qbc-SNARE synaptosome-associated protein 29) and the lysosomal factor VAMP8 (R-SNARE vesicle-associated membrane protein 8), as well as BRUCE, an inhibitor of apoptosis (IAP) protein, which is also involved in autophagosome/lysosome fusion (4,5).

Syntaxin 17 promotes initiation of PINK1/Parkin-independent mitophagy, which is regulated by depletion of the mitochondrial outer membrane protein Fis1 (6).

Phosphorylation of Syntaxin 17 at Ser202 by TBK1 promotes the formation of the mammalian pre-autophagosomal structure (mPAS) and interaction with core components Atg13 and FIP200.

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
  3. Levine, B. and Yuan, J. (2005) J Clin Invest 115, 2679-88.
  4. Viret, C. and Faure, M. (2019) Trends Cell Biol 29, 1-3.
  5. Corona, A.K. and Jackson, W.T. (2018) Trends Cell Biol 28, 869-81.
  6. Xian, H. et al. (2019) Nat Commun 10, 2059.
  7. Kumar, S. et al. (2019) Dev Cell 49, 130-144.e6.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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