All PLA reagents were purchased from Duolink (Sigma-Aldrich, St. Louis, MO, USA). PLA was performed according to manufacturer’s instructions. Briefly, differentiating cells from different time point (0-day ESCs, 1-day and 4-day EBs) of Strip2−- and Strip2+ESCs were fixed with 4% paraformaldehyde, incubated with PBS containing 0.1% Triton X-100, and blocked with Blocking Solution for 60 min. Cells were incubated with primary antibodies (TRIM28 and Strip2, ZFP57 and Strip2) at 4 °C overnight. After washing the cells three times with PBS-T, cells were incubated with oligonucleotide-conjugated secondary antibodies (PLA Probes) PLUS and MINUS for 1 h at 37 °C. Followed by washing with PBS-T and incubated with a ligation mixture (Detection Reagent Red) for 30 min at 37 °C. Cells were washed and incubated with an amplification mixture (Detection Reagent Red) for 100 min at 37 °C. After amplification, cells were washed and rinsed in distilled water. The nuclei were stained with DAPI. Images were captured with an inverted fluorescence microscope (Axiovert 200; Zeiss).
Pla reagents
Manufactured by Merck Group
Sourced in United States
PLA reagents are a set of laboratory chemicals used in the Proximity Ligation Assay (PLA) technique. PLA is a method for detecting and quantifying protein-protein interactions and post-translational modifications in biological samples. The PLA reagents facilitate the key steps of the PLA workflow, allowing researchers to analyze the presence, location, and abundance of specific protein complexes in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using pla reagents
1
Proximity Ligation Assay for Protein Interactions
2
Proximity Ligation Assay for HSV-1 Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PLA reagents were purchased from Sigma and the experiments were performed as previously described.57 (link) Briefly, cells were seeded on Teflon coted glasses and cultured for 20 h. After HSV-1 infection for 4 h, cells were f fixed in 4% paraformaldehyde for 10 min and washed with PBS for three times. After that, cells were fixed with 0.1% TritonX-100 on ice for 5 min, washed in PBS and blocked in 5% BSA in PBS for 1 h at 37 °C and incubated in primary antibodies overnight at 4 °C. On the next day, cells were washed with the wash buffer (DUO82049) for three times and incubated with the secondary antibodies with PLA probes (DUO92001-100RXN, DUO92005-100RXN) for 2 h at 37 °C followed by three times wash with the wash buffer. Cells were incubated with the Duolink in situ detection reagents red (DUO92008) for 2 h at 37 °C. Finally, cells were stained with the In Situ microplate nuclear stain and anti-fade (DUO82064-1KT) and covered onto slides. The images were acquired by using a confocal microscope (LSM 880, Carl Zeiss Microscopy Plan-Apochromat 40×/0.95 Korr M27) and the positive signals in the acquired images were quantified with Image-Pro Plus software and calculated as pixels per cell.
3
Proximity Ligation Assay for PI(4)P-ATM Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded on coverslips and, after the pertinent treatment, fixed with 4% PFA/PBS during 20 min at room temperature. Subsequently, every step took place in a humid chamber using the PLA reagents from Sigma‐Aldrich. Cells were permeabilized with 0.5% Triton/PBS for 10 min and incubated with Blocking Solution from the Duolink in situ PLA kit (DUO92013) for 1 h. Primary antibodies (anti‐PI(4)P and anti‐ATM) were diluted at 1:2,000 in the Antibodies Diluent from the kit and incubated overnight at 4°C onto the coverslips. For technical controls, one of these primary antibodies was omitted at a time. PLA minus and plus probes (DUO92004 and DUO92002, Sigma‐Aldrich) were next mixed and incubated in the Blocking Solution for 20 min as specified by the supplier, then added onto the coverslips, and incubated for 1 h at 37°C. Coverslips were washed twice with Buffer A (150 mM NaCl; 10 mM Tris; 0.05% Tween‐20; pH 7.4), then incubated with Ligation Mix (1:40 ligase; 1:5 ligase buffer) 30 min at 37°C, washed again twice with Buffer A, and then incubated with Polymerization Mix (1:80 polymerase; 1:5 polymerase buffer) 1 h at 37°C. Last, coverslips were washed with Buffer B (100 mM NaCl; 200 mM Tris; pH 7.5) and then with 0.01× Buffer B. The coverslips were finally mounted with Duolink in situ Mounting Medium with DAPI (DUO82040) and then sealed with nail polish.
4
Proximity Ligation Assay for HSV-1 Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PLA reagents were purchased from Sigma and the experiments were performed following the manufacturer’s instructions. Briefly, MEFs were seeded on Teflon-coated glasses and cultured for 18 h. After HSV-1 infection or transfection of HSV120 for 4 h, cells were fixed with 4% paraformaldehyde for 15–20 min at room temperature followed by permealization with 0.1% TritonX-100 for 10 min. Cells were blocked in 5% BSA in PBS for 30 min and incubated in primary antibodies for 2 h. Cells were washed with wash buffer A (DUO82049) for 3 times and incubated with the secondary antibodies with PLA probes (DUO92004-30RXN, DUO92002-30RXN) for 2 h at 37 °C followed by 3 times wash with the wash buffer A. The cells were then incubated with the Ligation-Ligase solution (DUO92008) for 30 min at 37 °C followed by 3 times wash with the wash buffer A. Cells were incubated with Amplification-Polymerase solution (DUO92008) for 100 min at 37 °C followed by 3 times wash with the wash buffer B (DUO82049). The nuclei were stained with DAPI for 2 min and then washed with PBS for 3 times. Imaging of the cells was carried out using Nikon A1 MP confocal microscope under a ×60 oil objective.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!