CD33 Rabbit Recombinant mAb

目录号：A5158

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20ul	447.89	现货
100ul	1500.13	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

MW (kDa)

65-75kDa

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	CD33 Rabbit Recombinant mAb detects endogenous level of CD33.
背景	CD33 is a myeloid specific member of the sialic acid-binding receptor family and is expressed highly on myeloid progenitor cells but at much lower levels in differentiated cells. Its expression is not seen in the earliest pluripotent progenitor cells, but it is expressed highly on cells committed to the myeloid lineage. CD33 expression is down-regulated with development of the myeloid lineage, resulting in low-level expression on peripheral granulocytes and tissue macrophages. Human CD33 has two tyrosine residues in its cytoplasmic domain (Y340 and Y358). When phosphorylated, these tyrosines could function as docking sites for the phosphatases, SHP-1 and/or SHP-2, enabling CD33 to function as an inhibitory receptor. CD33 belongs to a growing family of sialic acid-binding, immunoglobulin-like lectins (siglecs). It has two immunoglobulin-like domains and two cytoplasmic tyrosines. After phosphorylation, CD33 is capable of recruiting the protein tyrosine phosphatases SHP-1 and SHP-2, then negatively regulate cell activation.

特异性

CD33 Rabbit Recombinant mAb detects endogenous level of CD33.

背景

CD33 is a myeloid specific member of the sialic acid-binding receptor family and is expressed highly on myeloid progenitor cells but at much lower levels in differentiated cells. Its expression is not seen in the earliest pluripotent progenitor cells, but it is expressed highly on cells committed to the myeloid lineage. CD33 expression is down-regulated with development of the myeloid lineage, resulting in low-level expression on peripheral granulocytes and tissue macrophages. Human CD33 has two tyrosine residues in its cytoplasmic domain (Y340 and Y358). When phosphorylated, these tyrosines could function as docking sites for the phosphatases, SHP-1 and/or SHP-2, enabling CD33 to function as an inhibitory receptor. CD33 belongs to a growing family of sialic acid-binding, immunoglobulin-like lectins (siglecs). It has two immunoglobulin-like domains and two cytoplasmic tyrosines. After phosphorylation, CD33 is capable of recruiting the protein tyrosine phosphatases SHP-1 and SHP-2, then negatively regulate cell activation.

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] Paul SP, et al. Blood. 2000, 96(2):483-90.

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