Proximity ligation assays for sensitive and specific protein analyses

Gústafsdóttir, Sigrún M.; Schallmeiner, Edith; Simon, Fabrice; Gullberg, Mats; Söderberg, Ola; Jarvius, Malin; Jarvius, Jonas; Howell, Mathias; Landegren, Ulf

doi:10.1016/j.ab.2005.01.018

Cited by 113 publications

(109 citation statements)

References 32 publications

Supporting

Mentioning

109

Contrasting

Order By: Relevance

“…This was shown by PLA, which is a modern technology to study protein-protein interactions. 32,33 PLA has been used in the past to detect parvovirus and an intracellular bacterium 34 and our data indicate that it may become an important tool to investigate and verify binding of native and modified viruses and other pathogen to cell surface molecules. The PLA assay also revealed that insertion of FWKT motif in the HI loop allowed for binding to SSTR 2 but did not completely abolish CAR binding.…”

Section: Oncolytic Adenovirus With Somatostatin Motifs J Leja Et Almentioning

confidence: 90%

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Leja

Nilsson

et al. 2011

Gene Ther

View full text Add to dashboard Cite

We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackieadenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR 2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systemic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.

show abstract

Section: Oncolytic Adenovirus With Somatostatin Motifs J Leja Et Almentioning

confidence: 90%

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Leja

Nilsson

et al. 2011

Gene Ther

View full text Add to dashboard Cite

show abstract

“…Another notable technique involved aptamer-coordinated proximity ligation followed by PCR amplification and real-time detection, providing both high sensitivity and selectivity. (24,25) These three approaches exemplify the utility of combining DNA aptamers with PCR for protein target detection but rely on additional steps either before or after PCR for detection.…”

Section: Introductionmentioning

confidence: 99%

Protein detection via direct enzymatic amplification of short DNA aptamers

Fischer

Tarasow

Tok

2008

Analytical Biochemistry

View full text Add to dashboard Cite

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM).

show abstract

“…Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity [2][3][4] . This new technology couples antibody recognition with the amplification of DNA surrogate of the protein.…”

mentioning

confidence: 99%

Detection of Signaling Effector-Complexes Downstream of BMP4 Using <em>in situ</em> PLA, a Proximity Ligation Assay

Thymiakou

Episkopou

2011

JoVE

View full text Add to dashboard Cite

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1 . Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity [2][3][4] . This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation. Video LinkThe Fixation and Permeabilization1. Prepare the fixative, 4% PFA. Weigh 4 g of PFA to prepare a 4% solution in 100 ml PBS. Warm the solution at 50°C until it is clear. Filter the solution through a 0.22 μm filter and store at 4°C until use. Use fresh fixative (not stored for more than 2 days). 2. Aspirate the medium from the wells and wash with 1xPBS. Do not pipette the solutions directly onto the cells as this may result in detachment of the cells. Before going to the next step, if chamber slides are used remove the chambers, but leave the silicon around the wells. 3. Add 50 μl 4% PFA and incubate for 10min, at RT, without agitation. 4. Wash the cells with PBS for 3 x 5 min in a Coplin jar with agitation at RT. 5. Treat the cells with 0,5% Triton X-100 in PBS for 10 min without agitation at RT. 6. Wash the cells with 0,05% Tween 20 in TBS (TBS-T) for 3 x 5 min in a Coplin jar with agitation at RT.

show abstract

Proximity ligation assays for sensitive and specific protein analyses

Cited by 113 publications

References 32 publications

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Protein detection via direct enzymatic amplification of short DNA aptamers

Detection of Signaling Effector-Complexes Downstream of BMP4 Using <em>in situ</em> PLA, a Proximity Ligation Assay

Contact Info

Product

Resources

About