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- Table of Contents
Facts about Protein AMBP.
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Human | |
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Gene Name: | AMBP |
Uniprot: | P02760 |
Entrez: | 259 |
Belongs to: |
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No superfamily |
A1M; alpha 1Microglobulin; alpha 1-Microglobulin; alpha-1-microglobulin/bikunin precursor; AMBP; bikunin; EDC1; HCP; HCPcomplex-forming glycoprotein heterogeneous in charge; HI30; IATIL; inter-alpha-trypsin inhibitor light chain; ITI; ITIL; ITILC; ITILgrowth-inhibiting protein 19; protein AMBP; protein HC; trypstatin; uristatin; uronic-acid-rich protein; UTI
Mass (kDA):
38.999 kDA
Human | |
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Location: | 9q32 |
Sequence: | 9; NC_000009.12 (114060127..114078300, complement) |
Expressed by the liver and secreted in plasma. Alpha-1-microglobulin occurs in many physiological fluids including plasma, urine, and cerebrospinal fluid. Inter-alpha- trypsin inhibitor is present in plasma and urine.
Secreted.
Boster Bio's antibodies are high-affinity primary antibodies that have been extensively cited and proven in the research community. They are validated for use in Western Blotting, Immunohistochemistry, and ELISA. The AMBP marker is a specific subunit of the human albumin gene. Here are the best uses for the AMBP marker in your research. If you're looking for a high-affinity antibody to AMBP, Boster has the answer.
The Boster Bio AMBP marker is a highly specific and sensitive protein stain. It can be used to identify the presence of proteins in cell membranes. It has been validated for various detection methods, including Western Blotting, Immunohistochemistry, and ELISA. Membrane staining with this marker is particularly useful for detecting a wide range of proteins, including glycoproteins.
The AMBP marker is highly sensitive and is suitable for rapid protein band staining on nitrocellulose or PVDF membranes. The Ponceau S solution contains 0.2% w/v Ponceau S and 5% glacial acetic acid. The Ponceau S stain can be removed from membranes by repeatedly washing in wash buffer. The alkali treatment of the membrane prior to staining increases the signal. The solution must be prepared immediately before use. The nitrocellulose and PVDF membranes can be stained with a black band on a light background.
The Boster Bio product line specializes in IHC-optimized polyclonal antibodies and picogram-sensitivity ELISA kits. It offers over 12,000 antibodies, which have been thoroughly tested against known amounts of recombinant proteins, tissues, and un-transfected cell lines. Boster Bio antibody products are compatible with most major immunostaining methods, including IHC, WB, and Flow.
After the membranes were stained, they were cleaned with TBST (5 min) and incubated with enzyme-labelled goat anti-rabbit IgG antibody for 1 h at room temperature. Binding was visualized using ECL Plus reagents (Beyotime). Next, nitrocellulose membranes were fixed with a 50:50 methanol/water solution at 0 degC.
Human serum proteins were separated by 10% SDS-PAGE and transferred to PVDF or nitrocellulose membranes. These membranes were cut into five pieces and fixed with acetone or 50% methanol. After that, the samples were stained with LCA and SNA lectins and analyzed using quantitative methods. Three separate experiments were performed and the data were averaged across all of them.
The PVDF or nitrocellulose membranes used in this method are resistant to electroblotting, improving the retention of proteins. The PVDF membrane fixation improves IgG signal intensity. IgG is a low-molecular-weight protein. Heating the membranes at 50 degC or 100 degC for 30 min improved signal intensity. For western blotting, the Coomassie Brilliant Blue R-250 dye was used.
There are several methods to detect antibodies on the membrane. Indirect detection involves using a secondary antibody labeled with a biotin, fluorophore, or detectable enzyme. The secondary antibody is then attached to a measurable tag or colored product. This process detects the presence of the primary antibody and the antigen. Detection is possible if the antibody binds to the antigen.
The first method uses primary antibodies to stain the membrane. These antibodies bind to a specific protein, called the antigen. Secondary antibodies then recognize the first antibody bound to the target protein. Secondary antibodies are often linked to reporter enzymes that produce signals when they react with specific substrates. Secondary antibodies are specific to a particular species. When the antibodies are labelled with their specific epitope, they bind to that protein, thereby detecting the antigen.
In addition to direct detection of antibodies, Western blotting also involves indirect detection of antibodies. The first step of this technique is gel electrophoresis, either native or SDS-PAGE. Then, the separated macromolecules are transferred onto a membrane. The membrane will bind any protein, so it is important to block the membrane with a common protein before it is transferred. Detection methods vary widely, but most laboratories use indirect detection, in which the primary antibody binds to the target antigen, and the secondary antibody recognizes it.
Another method for detecting antibodies is immunofluorescence, or ANA, a technique that enables the detection of multiple specific ANAs. In immunofluorescence, the antigens that bind to the beads are conjugated with fluorescent dyes. The fluorescent light then reveals the antigens when the unbound reagent is washed off the slide. By comparing the resulting results to a positive control strip, the antibody-antigen complex is detected.
Zones of detection are the lines that form on the membrane when a fluid contains a specific antigen and analyte binds to the antigen-specific antibody. A visible line indicates that the test is working as desired, but an absent line means that the experiment is invalid. Detection of antibodies on the membrane requires a good amount of antigen-specific antibody titers and blocking agents.
To properly prepare the membrane for rapid immunodetection, the membrane must be thoroughly dried. To do this, refer to 'Membrane Drying Methods' and avoid rewetting with methanol. Then, diluted primary and secondary antibodies should be added to dilution buffer. The primary antibody solution should cover 0.1 mL per cm2 of the membrane. After one hour, the antibodies should move across the membrane's surface.
There are several methods to strip the membranes. One method involves washing the membranes in a low pH glycine-HCl buffer. A more aggressive method involves heating the membranes at high temperatures for 20 to 30 minutes. This method removes some of the transferred proteins, but may not completely remove the antibody. If you want to remove a large amount of transferred protein, use a high-pH solution of sodium dodecyl sulfate.
Another method is to add a small amount of conjugated secondary to the substrate, and observe the expected reaction. The substrate must also be active and the ECL reagent must be active. In photography, the x-ray film must be developed, and the film must be exposed to light for the final result. However, if the signal of the protein of interest is overpowered by the background, the primary antibody or its conjugate must be removed.
Before removing the primary antibody, it is essential to check its specificity. The primary antibody recognizes a specific epitope on the target protein, while the secondary antibody provides a means for detection. Secondary antibodies can be tagged with fluorescent or enzymes to produce color. A colorimetric assay can be documented by photocopy, film, or digital imager. The secondary antibody system is used for immunological research.
To use the same antibody for multiple Western blots, a nitrocellulose membrane is best. For dilutions of different antibodies, cut the membrane into 1 cm-wide strips. Label the strips with pencils. Another method is using PVDF membranes. Transfer the protein sample from the gel electrophoresis to the PVDF membrane. Make sure not to allow the membrane to dry.
PMID: 2428011 by Traboni C., et al. Sequence of a full length cDNA coding for human protein HC (alpha 1 microglobulin).
PMID: 2430261 by Kaumeyer J.F., et al. The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-alpha-trypsin inhibitor also encodes alpha-1-microglobulin (protein HC).