Affinity Ligands Design and Protocols

Introduction to Affinity Ligands

Affinity Ligands Applications

Affinity Ligands Design/Protocol

Affinity Ligands Literature

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Affinity Ligands Design / Protocol

I. Indirect Detection of Targets (In situ hybridization (ISH))

A. Principle challenges of ISH:

The mRNA target in a tissue or cell is typically at a lower concentration than that used in blotting work.

The mRNA target(s) may be masked by associated proteins.

The mRNA target(s) may be sequestered within a cell or cellular structure.

Successful probing of a tissue or cells for a particular mRNA requires (a) increasing cell permeability, and (b) increasing the visibility of the target mRNA sequence to the probe while preserving the tissue or cell�s structural integrity, and (c) to design the probe to achieve high resolution at high stringency.

B. ISH protocols�things to consider:

1. Preparation of Biological Material:

Frozen tissue sections?

Paraffin-embedded tissue sections?

Cellular suspension?

Each format has its own particular advantages/disadvantages, and its own requirements for proper preparation. Always remember that since RNA is very sensitive to degradation, RNA handling protocols must be strictly observed.

2. Types of Probes/Advantages of Oligo Probes

Several different types of probes can be used, such as chemically synthesized DNA oligo probes (20-50 bases), ssDNA probes, dsDNA probes, and RNA probes (all 200-500 bases long and enzymatically synthesized). However, in most cases, well-designed, purified, chemically synthesized DNA oligo probes are not only cost-effective (both in materials and labor) and convenient to use, but provide a number of technical advantages. In particular,

they are very stable and not subject to RNase degradation.

they can be more specific than the longer probe types: can be designed to selectively recognize different members of closely related gene families by targeting the most variable DNA sequence within a gene family.

they penetrate tissue better, due to their small size.

they produce more reproducible results overall--every DNA oligo probe is identical.

incorporation of affinity labels at the 5�- and 3�-ends does not interfere with hybridization to their respective targets. To avoid disruption of hybridization, internal labeling should be considered only for longer probes (30-50 bases).

3. Increasing Cell Permeability and mRNA target visibility

Increasing cell permeability involves treating the tissue or cells with some combination of (a) incubation in 0.2N HCl for 20-30 min (which extracts proteins from membranes), and (b) addition of Triton X-100 or SDS detergent (which extracts lipids from membranes). Detergent treatment is typically used for whole cell or frozen tissue sections. Increasing �visibility� of the mRNA target is accomplished by brief treatment of the tissue or cells with Proteinase K, which digests proteins that may be bound to the mRNA.

4. Pre-hybridization treatment to lower background staining

Because the DNA oligo probe contains affinity ligands for indirect detection, pre-hyb treatment of the tissue or cell sample is done to lower background staining. This is particularly important if the system used to visualize the probe-mRNA target complex utilizes enzymes such as peroxidase or alkaline phosphatase. To neutralize the endogenous presence of such enzymes in the sample, substances like 1% H2O2 (to block peroxidases) or levamisole (to block alkaline phosphatases) are added to the pre-hybridization solution.

5. Hybridization

For efficient hybridization of probe to mRNA target, hyb buffer typically includes the following components:

volume/water excluder (e.g., dextran sulfate): absorbs water, reducing that available for dissolving DNA, which increases probe concentration, and hyb rates.

organic solvents (e.g., formamide, DTT): reduce thermal stability of Watson-Crick bonds, allowing lower hyb temp.

monovalent cations (e.g., NaCl): interact with phosphate backbone to decrease electrostatic repulsion between DNA strands, and increases hyb rates.

EDTA: removes free divalent cations from solution, which strongly stabilize duplex DNA (undesirably increase hyb temp).

6. Recommended Controls

Poly dT/housekeeping gene probes--verifies tissue/tissue prep quality

RNase treatment of tissue sections, then hybridization w/labeled anti-sense probe--verifies that probe is actually binding to mRNA

Hybridize labeled sense and anti-sense probes in parallel to tissue sections--verifies probe binding is sequence-specific

References

(1) Pinkel, D., Straume, T., Gray, J.W. Cytogenetic analyses using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA (1986), 83: 2934-2938.
(2) Grzybowski, J., Will, D.W., Randall, R.E., Smith, C.A., Brown, T. Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups. Nucleic Acids Res. (1993), 21: 1705-1712.
(3) Kessler, C., Holtke, H.J., Seibl, R., Burg, J., Muhlegger, K. Nonradioactive labeling and detection of nucleic-acids I: a novel DNA labeling and detection system based on digoxigenin-anti-digoxigenin ELISA principle (digoxigenin system). Biol. Chem. Hoppe-Seyler (1990), 371: 917-927.
(4) Bianconcini, A., Lupo, A., Capone, S., Quadro, L., et al. Transcriptional activity of the murine retinol binding protein gene is regulated by a multiprotein complex containing HMGA1, p54nrb/NonO, protein-associated splicing factor (PSF) and steroidogenic factor 1 (SF1)/liver receptor homologue 1 (LRH-1). Int. J. Biochem. Cell Biol. (2009), 41: 2189-2203.