Difference between revisions of "Template:Heidelberg/pages/ab/introduction"
| (4 intermediate revisions by one other user not shown) | |||
| Line 14: | Line 14: | ||
<div class="col-lg-6"> | <div class="col-lg-6"> | ||
<p class="basictext"> | <p class="basictext"> | ||
| − | Western blotting is a common analytic technique to detect and quantify proteins. Traditionally, primary and secondary antibodies are widely used for this purpose. Although nowadays tens of thousands of antibodies are available for Western blotting, dependency from antibodies also raises several limitations: | + | Western blotting (or Immunoblotting) is a common analytic technique to detect and quantify proteins. Traditionally, primary and secondary antibodies are widely used for this purpose. Although nowadays tens of thousands of antibodies are available for Western blotting, dependency from antibodies also raises several limitations: |
</p> | </p> | ||
<ul class="basictext"> | <ul class="basictext"> | ||
| Line 24: | Line 24: | ||
</ul> | </ul> | ||
| − | |||
| − | |||
| − | |||
</div> | </div> | ||
| − | <div class="col-lg-6"> | + | <div class="col-lg-6" style="margin-top:-30px;"> |
<div class="imagewrapper"> | <div class="imagewrapper"> | ||
<div class="imagewrapperheader"> | <div class="imagewrapperheader"> | ||
| Line 43: | Line 40: | ||
AptaBodies consist of an aptamer, which binds to an immobilized protein of interest, a poly-A linker coupled to a HRP-mimicking DNAzyme forming a G-quadruplex. | AptaBodies consist of an aptamer, which binds to an immobilized protein of interest, a poly-A linker coupled to a HRP-mimicking DNAzyme forming a G-quadruplex. | ||
</p> | </p> | ||
| + | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 49: | Line 47: | ||
<div class="row"> | <div class="row"> | ||
<div class="col-lg-12"> | <div class="col-lg-12"> | ||
| + | <p class="basictext"> | ||
| + | |||
| + | Having these limitations in mind, our idea was to introduce aptamers into Western blotting. We decided to develop an “AptaBody” as a new tool for protein detection. AptaBodies are short DNA oligos, which combine the capabilities from primary and secondary antibodies within one molecule. At its 5’-end the AptaBody consists of an aptamer, targeting a specific molecule. Through this aptamer, the AptaBody can bind to an immobilized protein blotted on a membrane (Fig. 1). | ||
| + | </p> | ||
<p class="basictext"> | <p class="basictext"> | ||
Main advantages arguing for the application of aptamers for Western blotting are: | Main advantages arguing for the application of aptamers for Western blotting are: | ||
| Line 57: | Line 59: | ||
</ul> | </ul> | ||
<p class="basictext"> | <p class="basictext"> | ||
| − | Using our MAWS software we are able to design aptamers containing nucleotide sequences that optimally target every specific molecule of interest. On its 3’-end, the AptaBody contains the sequence of the HRP-mimicking DNAzyme. This oligonucleotide forms a G-quadruplex <x-ref>wang_highly_2015 </x-ref> (Wang, Wang, & Huang, 2015). In the presence of potassium ions, hemin can bind to the G-quadruplex, which then catalyses the luminol-H<sub>2</sub>O<sub>2</sub> chemiluminescence (CL) reaction. These two functional parts of the AptaBody are fused together via a poly-A linker (Fig. 1). | + | Using our MAWS software we are able to design aptamers containing nucleotide sequences that optimally target every specific molecule of interest. On its 3’-end, the AptaBody contains the sequence of the HRP-mimicking DNAzyme. This oligonucleotide forms a G-quadruplex <x-ref>wang_highly_2015</x-ref> (Wang, Wang, & Huang, 2015). In the presence of potassium ions, hemin can bind to the G-quadruplex, which then catalyses the luminol-H<sub>2</sub>O<sub>2</sub> chemiluminescence (CL) reaction. These two functional parts of the AptaBody are fused together via a poly-A linker (Fig. 1). |
</p> | </p> | ||
</div> <!-- col-lg-12 --> | </div> <!-- col-lg-12 --> | ||
Latest revision as of 18:12, 19 November 2015
Introduction
Western blotting (or Immunoblotting) is a common analytic technique to detect and quantify proteins. Traditionally, primary and secondary antibodies are widely used for this purpose. Although nowadays tens of thousands of antibodies are available for Western blotting, dependency from antibodies also raises several limitations:
- antibodies are often quite expensive
- production of specific antibodies is time consuming
- even if antibodies are available they might not work very well for the protein of interest
- their applicability is limited to a subset of proteins with sufficiently high molecular weight and certain biochemical properties
- for many proteins no antibodies are available
AptaBodies consist of an aptamer, which binds to an immobilized protein of interest, a poly-A linker coupled to a HRP-mimicking DNAzyme forming a G-quadruplex.
Having these limitations in mind, our idea was to introduce aptamers into Western blotting. We decided to develop an “AptaBody” as a new tool for protein detection. AptaBodies are short DNA oligos, which combine the capabilities from primary and secondary antibodies within one molecule. At its 5’-end the AptaBody consists of an aptamer, targeting a specific molecule. Through this aptamer, the AptaBody can bind to an immobilized protein blotted on a membrane (Fig. 1).
Main advantages arguing for the application of aptamers for Western blotting are:
- AptaBodies are very cheap (see cost)
- They are easy and fast to get
Using our MAWS software we are able to design aptamers containing nucleotide sequences that optimally target every specific molecule of interest. On its 3’-end, the AptaBody contains the sequence of the HRP-mimicking DNAzyme. This oligonucleotide forms a G-quadruplex