MICA Mouse Monoclonal Antibody [Clone ID: AMO1]

CAT#: AM26571AF-N

MICA mouse monoclonal antibody, clone AMO1, Azide Free

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Specifications

Product Data
Clone Name AMO1
Applications ELISA, FC
Recommended Dilution Flow Cytometry: 10 μg/ml (final concentration).
ELISA: 1 µg/ml (capture antibody).
Not recomended for Western Blot or Immunoprecipitation.
Reactivities Human
Host Mouse
Isotype IgG1
Clonality Monoclonal
Immunogen MICA*01, MICA*04 and MICB*02 transfected P815 cells
Specificity

This antibody reacts with MICA. The epitope was mapped to the helical surfaces of the MIC α1 α2 platform domain.
Formulation PBS containing 50% Glycerol, pH 7.2
State: Azide Free
State: Liquid purified Ig fraction
Preservative: None
Concentration lot specific
Purification Protein A Agarose Chromatography
Conjugation Unconjugated
Storage Upon receipt, store undiluted (in aliquots) at -20°C.
Avoid repeated freezing and thawing.
Stability Shelf life: One year from despatch.
Database Link
Background MICA and MICB (Major Histocompatibility Complex class I Chain-related gene A and gene B) bind to the activating immunoreceptor NKG2D. NKG2D is ex pressed on NK (Natural Killer) cells, NKT cells, γδ T cells and CD8 + αβ T cells. Recognition of MICA and MICB by NKG2D is involved in tumor surveillance, immune responses to viral infections and autoimmune diseases. MICA and MICB are transmembrane glycoproteins that are distantly related to the MIC proteins, and they possess three extra-cellular Ig-like domains. And thus, MICA and MICB are closely related but are functionally indistinguishable. MICA and MICB molecules are highly glycosylated, and are detected as a smear band ranging from 65-75 kDa. It is reported that MICA and MICB are highly expressed in variant tumor cells, whereas normal cells express little. Tumor cells have been shown to shed and release MIC molecules from the cell surface. Therefore determination of soluble MIC (sMIC) levels provides valuable information for cancer staging, and sMIC in serum seems to be an indicator for systemic manifestation of malignancy rather than for local tumor extent.
Synonyms MHC class I polypeptide-related sequence A, MIC-A, PERB11.1
Note Protocol:

Flow Cytometric analysis for floating cells
We usually use Fisher tubes or equivalents as reaction tubes for all step described below.
1) Wash the cells 3 times with washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3].
2) Resuspend the cells with washing buffer (5x10e6 cells/mL).
3) Add 50 μL of the cell suspension into each tube, and centrifuge at 500 x g for 1 minute at room temperature (20~25°C). Remove supernatant by careful aspiration.
4) Add 20 μ L of Clear Back (human Fc receptor blocking reagent) to the cell pellet after tapping. Mix well and incubate for 5 minutes at room temperature.
5) Add 40 μL of the primary antibody at the concentration of as suggest in the APPLICATIONS diluted in the washing buffer. Mix well and incubate for 30 minutes at room temperature.
6) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration.
7) Add 30 μL of 1:100 FITC conjugated anti-mouse IgG diluted with the washing buffer. Mix well and incubate for 15 minutes at room temperature.
8) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration.
9) Resuspend the cells with 500 μL of the washing buffer and analyze by a flow cytometer.
(Positive controls for Flow cytometry; 293T, Jurkat, HeLa)

ELISA
1) Distribute 100 μL/well of the anti-MICA monoclonal antibody (AMO1) (1 μg/mL) diluted with PBS to each well.
2) Incubate it overnight at 4°C.
3) Add 100 μL/well of 15% BSA/PBS.
4) Incubate it for 1 hour at 37°C.
5) Wash the plates 4 times with PBS-T [0.05% Tween-20 in PBS].
6) Distribute 100 μL/well of the samples or the recombinant MICA standard (0~15 ng/mL) diluted with 7.5% BSA/PBS to each well.
7) Incubate it for 2 hours at 37°C.
8) Wash the plates 4 times with PBS-T.
9) Distribute 100 μL/well of the anti-MICA/B monoclonal antibody (BAMO3) (1 μg/mL) to each well.
10) Incubate it for 2 hours at 37°C.
11) Wash the plates 4 times with PBS-T.
12) Distribute 100 μ L/well of the 1:5,000 HRP-conjugated anti-mouse IgG2a diluted with 3.75% BSA/PBS to each well.
13) Incubate it for 1 hour at 37°C.
14) Wash the plates 6 times with PBS-T.
15) Distribute 100 μL/well of the tetra-methylbenzidine (TMB) containing solution.
16) Incubate it for 5~60 minutes. The condition for reaction may vary.
17) Distribute 100 μL/well of 1 MH2SO4 to each well and stop enzyme reaction.
18) After gentle mixing, determine the absorbance at 450 nm of each well by a spectrophotometer
Reference Data

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