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M8Sac71 antibody as well as preparation method and application thereof

An m8sac71, antibody technology, applied in the direction of anti-enzyme immunoglobulin, serum immunoglobulin, measuring device, etc.

Inactive Publication Date: 2018-10-23
INST OF BASIC MEDICINE OF SAMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how to prove the existence of these acetylations still needs to prepare specific antibodies against these sites

Method used

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  • M8Sac71 antibody as well as preparation method and application thereof
  • M8Sac71 antibody as well as preparation method and application thereof
  • M8Sac71 antibody as well as preparation method and application thereof

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Experimental program
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Embodiment 1M8

[0039] Preparation and detection of embodiment 1M8Sac71 antibody

[0040] 1. Materials and instruments:

[0041] (1) Purification buffer:

[0042] Disodium hydrogen phosphate (Na 2 HPO 4 .12H 2 O), 29.01g;

[0043] Sodium dihydrogen phosphate (NaH 2 PO 4 .2H 2 O), 2.964g;

[0044] Sodium chloride, 9.00g;

[0045] wxya 2 0 to 1 L, pH = 7.4.

[0046] (2) Conjugation buffer: Purification bu+5mM EDTA.

[0047] (3) 5 mg BSA, Sigma A1933.

[0048] (4) Sulfo-SMCC (5 mg / ml) Thermo Scientific #22322, 50 mg.

[0049] (5) Freund's complete adjuvant and Freund's incomplete adjuvant: Sigma products.

[0050] (6) A glass syringe (10ml) with a good seal.

[0051] (7) Surgical instruments: several sets of scalpels, scissors, and tweezers.

[0052] (8) Artery clips, blood collection tubes, syringes, etc.

[0053] (9) Anesthetic: Chloral hydrate (10%).

[0054] (10) ELISA related reagents: 0.1M Na 2 CO 3 (PH=9.6), TBST, etc.

[0055] (11) Goat anti-rabbit antibody labeled w...

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Abstract

The invention discloses an M8Sac71 antibody. A preparation method of the M8Sac71 antibody comprises the following steps: (1) synthesis of an MARCH8Sac71 peptide fragment: preparing the MARCH8Sac71 peptide fragment, wherein the amino acid sequence of the peptide fragment is shown as Ac-T(Sac)ITPSSQDICRICHCEGDC-NH2; (II), coupling of the MARCH8Sac71 peptide fragment with BSA (Bull Serum Albumin) orKLH (Keyhole limpet hemocyanin); (III) preparation of an antibody with an immune rabbit; (IV) purification of the antibody: performing negative screening on the obtained serum by using a non-acetylated peptide fragment, and performing positive screening by using the MARCH8Sac71 peptide fragment to obtain an antibody, namely, the M8Sac71 antibody. The specificity of the M8Sac71 antibody is proved through dot-blot and competitive experiments, great rise of the acetylation level of MARCH8 co-transformed with serine acetylase YopJ is further proved, and the effectivity of the M8Sac71 antibody is confirmed. The M8Sac71 antibody can be used for detecting whether a 71-bit serine locus on the MARCH8 is acetylated or not.

Description

technical field [0001] The invention relates to a serine acetylated antibody, in particular to an antibody against MARCH8 whose 71-position serine site is acetylated, and a preparation method and application thereof. Background technique [0002] MARCH8 is a membrane-associated E3 ubiquitin ligase composed of 291 amino acids, which can target MHC II (HLA-DP, DQ, DR, HLA-DO, HLA-DM)], co-stimulatory molecule CD86 (B7-2 ) and CD8139], adhesion molecule CD44, apoptosis-related molecule Fas (CD95), Trail R, cell transport molecule TfR, IL-1 receptor accessory protein and other transmembrane molecules, through ubiquitination, endocytosis and lysosome The degradation pathway downregulates surface presentation of these molecules, regulating antigen presentation, T cell activation, apoptosis, and cellular trafficking. It is known that serine (S) at position 71 and position 75 and tyrosine (Y) at position 237 are phosphorylation sites, and lysine (K) at position 252 is a ubiquitinat...

Claims

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Application Information

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IPC IPC(8): C07K16/40C07K16/06G01N33/573
CPCC07K16/06C07K16/40G01N33/573G01N2333/9015
Inventor 李翠玲李冰清岳盈盈宋楠楠林玮王玮玮
Owner INST OF BASIC MEDICINE OF SAMS
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