PC9/p53EV, PC9/p53WT, and PC9/p53R248Q cells were cultured to 70% confluence on 12-mm-diameter coverslips (Matsunami, Osaka, Japan) placed in 24-well plates (Corning, Corning, NY). PC9/p53R248Q cells were treated with either DMSO as a control, 600 nM osimertinib, or TNF-α (1 ng/ml) for 24 h. Cells were fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 10 min with 0.3% Triton X-100 in PBS, and incubated overnight at 4 °C with 1:500 dilutions of mouse antibodies to p53 (#2524, Cell Signaling Technology) and rabbit antibodies to p65 (#8242, Cell Signaling Technology) for detection of p53-p65 complexes with the use of Duolink PLA Fluorescence Kits (#DUO92002 and #DUO92004; Sigma-Aldrich, St. Louis, MO, USA). Images of PLA signals were obtained with a BZX800 all-in-one fluorescence microscope (Keyence, Osaka, Japan). Optical sectioning images of p53-p65 complexes in multiple cells acquired from top to bottom of each cell were combined into a z-projection image with the use of full-focus imaging (BZ-H4A, Keyence). The number of PLA signals per cell and percentage of the signals in the nucleus stained with DAPI were quantified in nine fields including a total of at least 50 cells for each condition with the use of BZ-X Analyzer software (BZ-H4A, Keyence).
Duolink pla fluorescence kit
Manufactured by Merck Group
Sourced in United States
The Duolink® PLA Fluorescence kit is a laboratory tool designed for the detection and visualization of protein-protein interactions. The kit utilizes proximity ligation assay (PLA) technology to enable the specific and sensitive detection of these interactions in fixed cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti gfp, by Roche (2 mentions)
Rabbit anti gfp a 6455, by Thermo Fisher Scientific (2 mentions)
All in one fluorescence microscope bz x800, by Keyence (1 mentions)
24 well plate, by Corning (1 mentions)
12 mm diameter coverslips, by Matsunami (1 mentions)
Bz x analyzer software bz h4a, by Keyence (1 mentions)
Bz h4a, by Keyence (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Image analysis system, by Zeiss (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Carl lsm 700, by Zeiss (1 mentions)
Lsm780 confocal microscope system, by Zeiss (1 mentions)
Ab18197, by Abcam (1 mentions)
Pla0079, by Merck Group (1 mentions)
Inverted sp5x confocal microscope system, by Leica (1 mentions)
12 protocols using duolink pla fluorescence kit
1
Visualizing p53-p65 Complexes in NSCLC
2
Probing Protein Interactions via Duolink PLA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Interaction between HSP60 and RPTOR in cell cultures was probed using Duolink® PLA Fluorescence Kits (Sigma-Aldrich). In brief, formaldehyde-fixed cell cultures were blocked using Duolink® Blocking Solution and mixed with HSP60 and RPTOR antibodies, and secondary antibody Duolink® PLA probe. Specimens were incubated in a Duolink® ligation buffer containing 1 U/μl ligase at 37 ℃ for 30 min and followed by reacting with an amplification solution containing 2 U/ml polymerase and counterstaining with DAPI. Fluorescent spots in cell cultures were detected using a Zeiss fluorescence microscope, and number of fluorescent spots were counted using image analysis system (Carl Zeiss, Gottingen, Germany).
3
Proximity Ligation Assay for PfGRP170-BiP Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Asynchronous PfGRP170‐GFP‐DDD parasites were fixed as described above, approximately 24 hr after the removal of TMP. The PLA was performed using the Duolink PLA Fluorescence kit (Sigma) per the manufacturers protocol. For the BiP/PfGRP170 PLA assay, primary antibodies mouse anti‐GFP (Roche 11814460001, 1:500) and rabbit anti‐BiP MRA‐1246 (BEI resources (1:100) were used. For the negative control primary antibodies mouse anti‐plasmepsin V (from D. Goldberg, 1:1) and rabbit anti‐GFP A‐6455 (Invitrogen, 1:200) were used.
4
Visualizing iRhom2 Protein Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T iRhom1/2 DKO cells expressing N-terminal HA-tagged iRhom2 under tetracycline-inducible promoter were plated on 13-mm glass coverslips in 12-well plates and were treated with 250 ng/ml of doxycycline for 24 h to induce iR2 prior to fixation. Cells were washed 2 times with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. Cells were then washed 3 times with PBS and permeabilised in 0.3% TX-100 in PBS for 20 min. Coverslips were processed using Duolink® PLA Fluorescence kit (Sigma Aldrich, #DUO921101) according to manufacturer’s protocol. Images were acquired with a laser scanning confocal microscope (Fluoview FV1000; Olympus) with a 60 × 1.4 NA oil objective and processed using Fiji (Image J).
5
Proximity Ligation Assay for PfGRP170-GFP
6
Proximity Ligation Assay for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SKOV3 cells (4 × 104) were seeded onto sterilized cover slips and transfected with PIK3R1 siRNA. Interaction signals detected by proximity ligation assay were performed according to the instruction of Duolink PLA fluorescence kit (Sigma-Aldrich). Briefly, cells were fixed by 4% paraformaldehyde, followed by 0.1% Triton X-100 permeabilization and blocking. Next, cells were incubated with a mixture of two primary antibodies overnight. As negative control, one of the antibodies would be replaced by IgG. Then, the cells were incubated with PLA probe (PLUS and MINUS) for 1 h. Probe ligation was performed with two circle forming DNA nucleotides by ligation-ligase solution. PLA signals were amplified using amplification-polymerase solution. Slides were mounted with coverslip in the mounting medium with DAPI. Fluorescence signals were observed at 461 nm (DAPI) and 594 nm (Texas Red) by Carl Zeiss LSM 700 (Zeiss). For each set of samples, at least three fields were captured and spots per nucleus were counted. Experiments were performed at least three times independently.
7
Proximity Ligation for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proximity ligation assay was performed using the Duolink PLA Fluorescence Kit (Sigma) according to the manufacturer's instructions. U2OS cells were subjected to a ultraviolet-A laser (λ = 355 nm, 40% energy) and collected at different time points after microirradiation. Cells were fixed in 4% paraformaldehyde for 10 min, and permeabilized with PBS containing 0.2% Triton X-100 for 10 min at room temperature. Cells were incubated with Duolink blocking solution at 37°C for 1 h followed by primary antibodies (anti-USP11 and anti-MTA2; anti-USP11 and anti-HDAC2) over night at 4°C. Then, cells were incubated with proximity ligation assay probes PLUS and MINUS for 1 h at 37°C and ligation–ligase solution for 30 min at 37°C. After ligation, cells were incubated with an amplification polymerase solution for 100 min at 37°C. Cells were counterstained for 1 h at room temperature with secondary antibodies coupled to Alexa Fluor 594 to stain MTA2 or HDAC2, and a final concentration of 0.1 μg/ml DAPI was included to stain nuclei. Images were acquired with a LSM880 laser scanning confocal system.
8
Visualizing Protein Interactions via Duolink PLA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In situ protein-protein interactions were detected using the Duolink PLA fluorescence kit (Sigma-Aldrich) according to the manufacturer's instructions. Primary antibodies included mouse anti-SRGN (#sc-393521, Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-MDK (#AF-258-PB, R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-MMP2 (#ab92536, Abcam, Cambridge, United Kingdom) and rabbit anti-MMP9 (#ab76003, Abcam). Briefly, SRGN and a potential interacting protein were detected with a pair of primary antibodies raised in different species, followed by oligonucleotide-labeled secondary antibodies (PLUS and MINUS PLA probes). Connector oligonucleotides were then applied to hybridize and join the probes that were in close proximity (< 40 nm apart) into a closed circular DNA template for rolling circle amplification in the presence of fluorescent-labeled oligonucleotides to form a distinct fluorescent spot 18 (link). Images were captured using a Carl Zeiss LSM 780 confocal microscope system (Zeiss, Jena, Germany).
9
Proximity Ligation Assay for S9.6-PCNA and RNAPII-PCNA Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the S9.6-PCNA PLA, cells were incubated in CSK buffer (10 mM PIPES, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA and 0.5% Triton X-100™) for 10 min at 4°C, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, blocked with 10% fetal bovine serum in PBS for 1 h at 37°C in a humidity chamber. For the RNAPII-PCNA PLA, cells grown on coverslips were washed with PBS, fixed with 4% PFA for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. Cells were then blocked in 3% BSA and 0.1% Tween 20 in 4× SSC for 1 h at room temperature. After blocking, cells were incubated with primary antibody overnight at 4°C (1:250 mouse anti-S9.6 antibody (ENH001, Kerafast) and 1:250 rabbit anti-PCNA antibody (ab18197, Abcam) for S9.6-PCNA PLA; 1:500 goat anti-RNA polymerase II antibody (PLA0292, Sigma) and 1:500 rabbit anti-PCNA antibody (PLA0079, Sigma) for RNAPII-PCNA PLA). The next day, after washing with 1× PBS twice, cells were incubated with pre-mixed Duolink PLA plus and minus probes for 1 h at 37°C. The subsequent steps in proximal ligation assay were carried out using the Duolink® PLA Fluorescence kit (Sigma) according to the manufacturer's instructions. Slides were then stained with DAPI and imaged on a Zeiss LSM880 confocal microscope for S9.6-PCNA PLA and Zeiss LeicaDM18 microscope for RNAPII-PCNA PLA.
10
Mapping RNA-DNA Interactions in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were transfected with FLAG tagged THOC5, THOC6 and empty vector for two days. Cells were fixed in 4% PFA for 15 min at RT and permeabilized with 0.2% Triton X-100 for 5 min. Samples were blocked in blocking buffer of Proximity ligation assay for 1h at 37°C. After blocking, samples were incubated with S9.6 and FLAG antibody (rabbit, Sigma) (1:200 dilution in antibody dilution buffer) for 2h at 37°C. After 2x washing with wash buffer A, the samples were incubated with pre-mixed Duolink PLA plus and minus probes for 1 h at 37°C. The subsequent steps in proximal ligation assay were carried out using the Duolink® PLA Fluorescence kit (Sigma) according to the manufacturer’s instructions.
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!