Cathepsin L/V/K/H Rabbit Recombinant mAb

目录号：A5284

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20ul	447.66	现货
100ul	1500.38	现货
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使用信息

抗体应用

WB,ELISA

稀释比例

1:1000

反应性

Human Mouse Rat

MW (kDa)

38kDa

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	Cathepsin L/V/K/H Rabbit Recombinant mAb detects endogenous level of total Cathepsin L/V/K/H.
背景	There are 11 human cysteine cathepsins, i.e., the cathepsins B, C, F, H, K, L, O, S, V, X and W. The majority of cathepsins are ubiquitously expressed in human tissues such as cathepsins B, H, L, C, X, F, O and V. Their expression profile indicates that these enzymes are involved in a normal cellular protein degradation and turnover. In contrast, cathepsins K, W and S show a restricted cell or tissue-specific distribution. Cathepsin K is highly expressed in osteoclasts, in most epithelial cells and in the synovial fibroblasts in rheumatoid arthritis joints. Cathepsin V (also named L2) is highly homologous to cathepsin L, but in contrast to the ubiquitously expressed cathepsin L, its expression is restricted to thymus and testis. Active cathepsins are also localized in other cellular compartments, such as the nucleus, cytoplasm and plasma membrane. Cysteine cathepsins exhibit broad specificity, thus cleaving their substrates preferentially after basic or hydrophobic residues. They are believed to participate exclusively in terminal protein degradation during necrotic and autophagic cell death. They also execute numerous specific functions participating in important physiological processes, such as MHC-II-mediated antigen presentation, bone remodeling, keratinocyte differentiation, prohormones activation, and others.

特异性

Cathepsin L/V/K/H Rabbit Recombinant mAb detects endogenous level of total Cathepsin L/V/K/H.

背景

There are 11 human cysteine cathepsins, i.e., the cathepsins B, C, F, H, K, L, O, S, V, X and W. The majority of cathepsins are ubiquitously expressed in human tissues such as cathepsins B, H, L, C, X, F, O and V. Their expression profile indicates that these enzymes are involved in a normal cellular protein degradation and turnover. In contrast, cathepsins K, W and S show a restricted cell or tissue-specific distribution. Cathepsin K is highly expressed in osteoclasts, in most epithelial cells and in the synovial fibroblasts in rheumatoid arthritis joints. Cathepsin V (also named L2) is highly homologous to cathepsin L, but in contrast to the ubiquitously expressed cathepsin L, its expression is restricted to thymus and testis. Active cathepsins are also localized in other cellular compartments, such as the nucleus, cytoplasm and plasma membrane. Cysteine cathepsins exhibit broad specificity, thus cleaving their substrates preferentially after basic or hydrophobic residues. They are believed to participate exclusively in terminal protein degradation during necrotic and autophagic cell death. They also execute numerous specific functions participating in important physiological processes, such as MHC-II-mediated antigen presentation, bone remodeling, keratinocyte differentiation, prohormones activation, and others.

实验方法

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.

参考文献	[1] Olson OC, et al. Nat Rev Cancer. 2015, 15(12):712-29.

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