HMGB1 Rabbit Recombinant mAb

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  • WB
  • IF
规格 价格 库存 购买数量
20ul 447.96 现货
100ul 1500.17 现货
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400-668-6834

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使用信息

抗体应用 WB, IF,ELISA
稀释比例
WB IF
1:1000 1:50
反应性 Human Mouse Rat
MW (kDa) 25kDa
抗体类型 Rabbit
浓度 1mg/ml
储存液配方 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
储存条件
(自收到货起)
Store at –20°C.

Datasheet & SDS

生物描述

特异性 HMGB1 Rabbit Recombinant mAb 可检测HMGB1内源性水平。
背景 HMGB1是一种进化上很古老的蛋白,最新发现它还是一种细胞因子。早期对HMGB1的研究主要集中在其DNA结合蛋白的作用上,它经常与染色质DNA一起被纯化出来。但它与染色质只是松散地结合,不像组蛋白一样与染色质紧密结合。HMGB1是一种核蛋白,存在于大多数真核细胞中,稳定核小体的形成并作为转录因子样蛋白调节基因的表达。在伤害、感染或其他炎症刺激下,活化的巨噬细胞、成熟树突细胞、自然杀伤细胞也可分泌HMGB1。HMGB1可引起DNA弯曲、帮助多种调节蛋白复合物结合DNA。它还有助于转座子的整合。HMGB1可增强其他蛋白与DNA的相互作用,增强转录激活。HMGB1介导系统性炎症反应,由活化免疫细胞分泌,激活免疫细胞和内皮细胞中典型的炎症反应,通过RAGE和其他受体(如TLR2、TLR4)转导细胞信号。它在不同的组织中具有特异活性。

实验方法

WB

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

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

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). 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.

IF

Immunofluorescence (Immunocytochemistry)

Specimen Preparation (forcultured cell lines, IF-IC)

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. Fix cells for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining.

Immunostaining

1. Add theblocking buffer and incubate for 60 min at RT.
2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
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 specimens 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. Mount slides usingmounting medium with DAPI and cover with coverslips.
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.

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