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2743
ISG15 Antibody
一抗
多克隆抗体

ISG15 Antibody #2743

Citations (72)
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  1. WB
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Western blot analysis of lysates from HeLa, A549, RAW and COS cells, treated with or without IFN (1000 U/mL) for 24 hours, using ISG15 antibody.
Western blot analysis of NEDD8, Ubiquitin, ISG15 and SUMO-2/3 recombinant proteins (5 ng each), using NEDD8 (#2745) , Ubiquitin (#3936), ISG15 (#2743) and SUMO-2/3 (#4974) Antibodies.
Flow cytometric analysis of Hela cells, untreated (blue) or IFNa treated (green), using ISG15 antibody compared to a nonspecific negative control antibody (red).
The relationship between recombinant ISG15 protein concentration and assay optical density readings. Recombinant NEDD8 protein was used as a negative control.
To Purchase # 2743S
Cat. # Size Price Inventory
2743S
100 µl

Supporting Data

REACTIVITY H M Mk
SENSITIVITY Endogenous
MW (kDa) 15
SOURCE Rabbit

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
Flow Cytometry (Fixed/Permeabilized) 1:200
Peptide ELISA (DELFIA) 1:100

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 BSA, 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

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.

  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. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. 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 (#59329, 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 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-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 June 2020

Protocol Id: 10

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

肽 ELISA

A. 溶液与试剂

  1. 碳酸盐缓冲液:15 mM Na2CO3、35 mM NaHCO3、0.2 g/L NaN3 (pH 9.6)。将 1 μM 合成肽溶于碳酸盐缓冲液中。
  2. 10X Phosphate Buffered Saline (PBS):要配制 1 L,添加 80​ g 氯化钠 (NaCl)、2 g 氯化钾 (KCl)、14.4 g 磷酸氢二钠 (Na2HPO4) 和 2.4 g 磷酸二氢钾 (KH2PO4) 到 1 L dH2O 中。调节 pH 至 7.4。
  3. 洗涤缓冲液:含 0.05% Tween-20 的 1X PBS (PBST)
  4. 封闭缓冲液:用 PBST 稀释的 10 mg/ml 牛血清白蛋白 (BSA)。
  5. 抗体稀释缓冲液:用 PBST 稀释的 3% BSA
  6. DELFIA® 铕标记的针对小鼠一抗的 Anti-mouse IgG 或针对兔一抗的 Anti-rabbit IgG (PerkinElmer Life Sciences #AD0124)。
  7. DELFIA® 增强溶液 (PerkinElmer Life Sciences #1244-105)

(DELFIA® 是 PerkinElmer, Inc. 的注册商标)

B. 实验步骤

  1. 96 孔微量滴定平板的各孔用通过碳酸盐缓冲液配制的 100 μl 1 μM 合成肽包被,在 4°C 下过夜孵育或在 37°C 下孵育 2-6 小时。如果肽不结合或不吸附,可尝试pH在4–8 内的其他缓冲液。
  2. 用 200 μl/孔的洗涤缓冲液洗涤平板 3 次。
  3. 在 37°C 下,平板按 200 µl/孔用封闭缓冲液封闭 1 小时。将洗涤缓冲液洗涤平板 3 次。(如果需要,可以持续 1-2 个月将平板保持在 4°C )
  4. 用抗体稀释缓冲液配制适宜稀释度的一抗 。向每个孔中添加 100 μl,并在 37°C 下孵育 1 小时。
  5. 用洗涤缓冲液洗涤 3 次。
  6. 按 67 ng/孔添加稀释于 100 μl/孔抗体稀释缓冲液中的 DELFIA 铕标记 Anti-mouse IgG 或 Anti-rabbit IgG。在轻柔振荡摇床上室温孵育 30 分钟。
  7. 用洗涤缓冲液洗涤 3 次。
  8. 添加 100 μl 增强溶液并在室温下孵育 5 分钟。用适宜的时间分辨平板读数仪在 615 nm 读取平板读数。

发布​时间 2005 年 6 月

修订时间 2007 年 9 月

实验步骤编号:34

特异性/灵敏度

该抗体可检测游离和偶联 ISG15 蛋白的内源水平。抗体不会与其他泛素家族成员发生交叉反应,包括泛素、SUMO1、SUMO2、SUMO3、NEDD8。

物种反应性:

人, 小鼠, 猴

来源/纯化

使用与源自人类 ISG15 蛋白氨基酸相对应的合成肽,对动物进行免疫接种来产生多克隆抗体。通过蛋白质 A 和肽亲和色谱纯化抗体。

背景

干扰素刺激 15 kDa 蛋白 (ISG15) 也称为泛素交叉反应性蛋白 (UCRP),是泛素样蛋白家族的成员之一,并在妊娠到天然免疫应答等多种生物学通路中发挥作用 (1)。ISG15 的表达受到暴露于细胞的 I 型干扰素 α 和 β 刺激,除此之外也可受到病毒感染的刺激,例如 (B) 型流感 (2,3)。暴露于 I 型干扰素后,除一些成纤维细胞核上皮细胞外,淋巴细胞和单核细胞也可释放 ISG15 到培养基 (1,4)。已证实 ISG15 作为细胞因子,可刺激自然杀手细胞的增殖、中性粒细胞的趋化效应,也可通过单核细胞和巨噬细胞刺激干扰素 γ 的分泌。 (4,5)。亦证实 ISG15 在细胞内发挥作用,通过 E1 (Ube1L)、E2 (UbcH8) 和 E3 连接酶经多种类似于泛素化作用的步骤,会与其他蛋白共价结合 (6,7)。ISG15 通过泛素化加工蛋白酶 Ubp43,与其它蛋白解离 (8)。ISG15-蛋白结合物 (ISGylation) 由 I 型干扰素诱导,并作用于包括丝氨酸蛋白抑制剂 Serpin 2A、PLCγ1、ERK1/2、Jak1 和 Stat1 等靶标蛋白 (9,10)。ISGylation 不同于泛素化作用,并不将蛋白靶标降解,相反 ISGylation 提高了 Jak1 和 Stat1 的活性,加强了对干扰素的细胞应答 (11)。

有限使用

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仅供研究使用。不得用于诊断流程。
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