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70973
Phospho-Ubiquitin (Ser65) (E5T1W) Rabbit mAb
一抗
单克隆抗体
R
Recombinant

Phospho-Ubiquitin (Ser65) (E5T1W) Rabbit mAb #70973

Citations (4)
Filter:
  1. IF
  2. F
Confocal immunofluorescent analysis of PC-3 cells, untreated (left), treated with CCCP (30 μM, 6 hr; center), or treated with CCCP and post-processed with λ-phosphatase (right), using Phospho-Ubiquitin (Ser65) (E5T1W) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue = Hoechst 33342 #4082 (fluorescent DNA dye).
Flow cytometric analysis of PC-3 cells, untreated (blue) or treated with CCCP (30 μM, 6 hr; green), using Phospho-Ubiquitin (Ser65) (E5T1W) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
To Purchase # 70973S
Cat. # Size Price Inventory
70973S
100 µl

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit IgG

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
Immunofluorescence (Immunocytochemistry) 1:1000
Flow Cytometry (Fixed/Permeabilized) 1:150 - 1:600

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.

For a carrier free (BSA and azide free) version of this product see product #43163.

Protocol

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit

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

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 24

Flow Cytometry, Methanol Permeabilization Protocol for Rabbit Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Rabbit secondary antibodies::
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
  7. Incubate for 30 min at room temperature. Protect from light.
  8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised June 2020

实验步骤编号:404

特异性/灵敏度

Phospho-Ubiquitin (Ser65) (E5T1W) Rabbit mAb 可检测仅在 Ser65 位点被磷酸化的nei'yuan内源水平的泛素蛋白。

物种反应性:

基于 100% 序列同源性预测发生反应的物种:

小鼠, 大鼠

来源/纯化

使用与人泛素蛋白中 Ser65 周围的残基相对应的合成磷酸肽对动物进行免疫接种来产生单克隆抗体。

背景

泛素是一个保守的多肽单位,在泛素-蛋白酶体通路中发挥重要作用。泛素可通过泛素化过程共价连接许多细胞蛋白,在这个过程中,26S 蛋白酶体降解靶标蛋白。三种组分与靶标蛋白-泛素接合过程有关。首先,泛素通过与激活组分 E1 形成一种硫酯复合体而被激活;激活的泛素继而转运至泛素载体蛋白 E2,然后从 E2 转运到泛素连接酶 E3,最后从 E3 转运到靶标蛋白赖氨酸残基的 ε-NH2 (1-3)。泛素-蛋白酶体通路与各种正常生物过程和疾病相关异常有关。IκB、p53、cdc25A 和 Bcl-2 等多种蛋白已被证实是泛素-蛋白酶体过程的靶标,可参与细胞周期进程、分化、细胞应激反应和凋亡的调节 (4-7)。

泛素会在 Ser65 位点被 PINK1 磷酸化,导致 E3 泛素连接酶 Parkin 被激活 (8, 9)。PINK1 在去极化的线粒体上聚集,从而磷酸化泛素蛋白并激活 Parkin,随后诱发线粒体自噬通路清除受损的线粒体。PINK1 和 Parkin 的功能丧失性突变会引起早发性帕金森病 (10, 11)。

有限使用

除非如以 CST 合法授权代表签署的书面形式另行明确同意,否则以下条款适用于 CST、其附属公司或其分销商提供的产品。除非 CST 合法授权代表以书面形式单独接受,否则任何附加于或异于此处所载条款和条件的客户条款和条件均被拒绝且无效。

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仅供研究使用。不得用于诊断流程。
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