Proteins in the human cells, believed to be more than total number of genes (>22,000) are the crucial molecules involved in maintaining integrity and normal functioning of the entire system. Study of proteins and their function is hence important for understanding the molecular mechanisms involved within and between the cells. Protein isolation and identification is the major step in studying their biological process within the system. This has been made possible through variety of techniques like chromatography, mass spectrometry, ELISA, western blot and still more. One such new approach for protein detection and identification is the proximity ligation assay (PLA) which was first described in the year 2002 by Fredriksson et al. In PLA, target proteins are detected using affinity molecules linked to oligonucleotide sequence called as PLA probes which upon their proximity are hybridized to a connecter oligonucleotide, enzymatically ligated and quantified upon DNA amplification. In PLA target proteins in solution are detected in a homogenous phase without any solid support for the target molecule whereas in solid phase PLA (sp-PLA), target molecules are initially bound to a solid support which enhances detection of low concentration proteins engaged in complex biological
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Using a solid support for capturing target molecules as in sp-PLA helps in concentrating target molecules and eliminating other interfering substances existing in samples. Also three antibodies targeting three different epitopes of a single protein renders sp-PLA a highly specific assay. Studies using sp-PLA has revealed it to be 100 fold greater sensitive than ELISA as it detects even low fM range of molecules with broader dynamic range up to 6 orders of magnitude. [1-4]
Vascular endothelial growth factor, VEGF is a signal protein produced from the cells that stimulate vasculogenesis and angiogenesis. As a result of alternative splicing VEGF mRNA encodes for six different isoforms of VEGF (VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, VEGF206). These isoforms differ in mainly their molecular weight and in properties like binding to cell surface heparan sulfate proteoglycans. Monomeric forms of isoforms tend to form active homodimers by disulfide bond formation. VEGF expression induces cell proliferation, cell migration upon binding to tyrosine kinase signaling receptors VEGFR1, VEGFR2 and VEGFR3 which ends up in angiogenic process (fig 2). VEGF also plays a centralized role in vasculogenesis (de novo blood vessel formation at embryonic developmental stage). In here, we have used solid phase proximity ligation assay (sp-PLA) with magnetic beads and anti-VEGF polyclonal antibody to determine the concentration of VEGF proteins in two
human serum samples (S1 and S2). [5,6]
[R.Rial et al.,(5)]
Abiarchana G, Krishnapriya L, Sindhuja P Page 2
Materials and Method
Two important steps to be carried out prior performing sp-PLA are: preparation of PLA probes and preparation of capture beads (solid support). PLA probes of
50nM are prepared by initially diluting the biotinylated anti-VEGF antibody (1ÂµM) with antibody dilution buffer (1X PBS and 0.1% BSA) in ration 1:10. Following antibody dilution, equal volume of diluted antibody is mixed with
100nM of each streptavidin conjugated oligonucleotide (SLC1 and SLC2) separately and incubated for 1 hr at room temperature (RT). Preparing PLA probes on separate tubes avoids loss of either of the probes (3â€™ free end probe, SLC1 and 5â€™ free end probe, SLC2) and also yields us equal amount of both probes efficiently.
Solid support, streptavidin coated magnetic beads with biotinylated capture antibody are prepared by following the steps as described. Beads are initially vortexed and washed twice with wash buffer (1X PBS and 0.05% Tween 20) using magnetic rack. Biotinylated anti-VEGF antibody diluted to 50nM was then added to washed beads and incubated at RT for 1 hr under rotation to make efficient binding of antibody to beads. Later, unbound antibodies are removed by washing the incubated capture beads twice with wash buffer. Capture beads could be stored using storing buffer if necessary.
VEGF proteins with known concentration prepared upon serial dilution from
1nM to 0.1pM (and 0pM as control) are used in the experiment to determine the concentration of VEGF from two unknown samples (S1 and S2). Prepared capture beads (storing buffer washed if present) with washing buffer replaced with assay buffer (1X PBS, 0.05% Tween 20, 0.1% BSA, 1mM biotin, 100nM IgG, 100Âµg/ml salmon sperm DNA) was added to each well in a 96 well plate [experiment was performed in triplicate (1nM-0pM, S1 and S2)* 3 hence 24 wells of 96 were used in total] followed by addition of diluted VEGF and unknown samples in ratio 1:9. Well plates were then firmly covered and incubated at RT under rotation for 1hr to make the beads homogeneous. Following incubation, well plates were briefly spun and washed twice with wash buffer to remove unbound VEGF. Separately prepared PLA probes were now diluted in ratio 1:100 in two different tubes and finally mixed in a single tube. Freshly mixed PLA probes of volume equal as that of diluted VEGF were then added to each well and incubated at RT for 1hr under rotation.
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From the cycle threshold (Ct) value obtained for each well after running a real time PCR was averaged for protein samples with different concentrations. From mean Ct value and known VEGF concentrations, a calibration curve was plotted as shown in figure 3. Solid phase PLA performed against VEGF proteins exhibited low limit of detection (LOD) which was in the range of pM. Also, it shows considerable dynamic range. All these observations coupled less background (observation from blank) suggests that even samples containing very low concentration of proteins could be detected and determined efficiently by performing sp-PLA. Concentration of VEGF proteins in two human serum samples was determined by using the mean Ct values obtained from RT-PCR on the equation obtained upon plotting calibration curve.