TCR-activated PBMCs employed for the MethylScan study were used to generate lysates as described above for Western blot analysis. Protein concentrations were determined using the BCA Protein Assay Kit. The anti-ADMA antibody (CST, clone D10F7A10) was biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific, #21326) according to the manufacturer’s instructions. 1 µg of lysates, in triplicates, was incubated with the biotinylated anti-ADMA antibody, at a concentration of 0.5 µg/mL, and antibodies specific to ALYREF (Abcam, #ab6141), DHX9 (Abcam, #ab54593), EIF4H (Abcam, #ab77455), EWSR1 (Abcam, #ab54708), G3BP1 (Abcam, #ab56574), HNRNPA1 (Abcam, #ab5832), KHDRSB1 (Abcam, #ab56836), SFPQ (Abcam, #ab11825) and TPR (Abcam, #ab58344) at a concentration of 1 µg/mL. After a 45-min incubation at room temperature, streptavidin-coated acceptor beads (Perkin Elmer, #AL125C) and anti-mouse IgG-coated donor beads (Perkin Elmer, #AS104) were added to respective wells of 96 well microplates (Corning, #3693), at a final concentration of 20 µg/mL and incubated for an additional 45 min at room temperature. All solutions were prepared in AlphaLISA universal buffer (Perkin Elmer, #AL001F) and microplates were shaken after each addition and centrifuged prior to luminescence detection on an Envision microplate reader (Perkin Elmer).
Al125c
Manufactured by PerkinElmer
The AL125C is a laboratory instrument designed for the analysis and quantification of various analytes. It is a compact, automated system that utilizes advanced spectroscopic techniques to provide accurate and reliable results. The core function of the AL125C is to perform precise measurements and data analysis for a wide range of applications in the scientific and research domains.
Automatically generated - may contain errors
Lab products found in correlation
Ab54708, by Abcam (1 mentions)
Ab54593, by Abcam (1 mentions)
Ab5832, by Abcam (1 mentions)
Ab11825, by Abcam (1 mentions)
Ab56574, by Abcam (1 mentions)
Ez link sulfo nhs biotin kit, by Thermo Fisher Scientific (1 mentions)
Envision microplate reader, by PerkinElmer (1 mentions)
Streptavidin donor bead, by PerkinElmer (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
2 protocols using al125c
1
Quantification of Arginine Methylation
2
AlphaLISA for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
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AlphaLISA assays were performed in Optiplate-384 plus plates using
Anti-GFP AlphaLISA Acceptor (AL133C, PerkinElmer, AUS) and Streptavidin
Donor beads (AL125C, PerkinElmer, AUS). α beads were prepared
according to the protocol provided by the manufacturer.53 Briefly, the acceptor and donor bead stocks
(5 mg/mL) were diluted to 100 μg/mL (5×) in AlphaLISA assay
buffer (Buffer A: 25 mM HEPES, 50 mM NaCl, 0.1% BSA and 0.01% Nonidet
P-40; pH: 7.5). The biotinylated mCherry nanobody (prepared in-house)23 diluted in buffer A (final concentration of
4 nM) was added into microplate wells, followed by the addition 15
μL of LTE lysate diluted 1:20 with assay buffer and containing
putative interacting proteins (diluted 20× in AlphaLISA assay
buffer) and 5 μL of the acceptor beads (5×). The samples
were incubated for 30 min at room temperature. Subsequently, 5 μL
of donor beads (5×) were added to samples under low light conditions
and incubated for 30 min at room temperature. For all experiments,
samples were prepared in triplicate and the assay was repeated 2 times.
The AlphaLISA signal was detected with the Tecan Spark multimode microplate
reader using the following settings: mode: AlphaLISA, excitation time:
130 ms, and integration time: 300 ms. In α screen experiments,
the FKBP-rapamycin binding (FRB) protein was used as a negative control.
Anti-GFP AlphaLISA Acceptor (AL133C, PerkinElmer, AUS) and Streptavidin
Donor beads (AL125C, PerkinElmer, AUS). α beads were prepared
according to the protocol provided by the manufacturer.53 Briefly, the acceptor and donor bead stocks
(5 mg/mL) were diluted to 100 μg/mL (5×) in AlphaLISA assay
buffer (Buffer A: 25 mM HEPES, 50 mM NaCl, 0.1% BSA and 0.01% Nonidet
P-40; pH: 7.5). The biotinylated mCherry nanobody (prepared in-house)23 diluted in buffer A (final concentration of
4 nM) was added into microplate wells, followed by the addition 15
μL of LTE lysate diluted 1:20 with assay buffer and containing
putative interacting proteins (diluted 20× in AlphaLISA assay
buffer) and 5 μL of the acceptor beads (5×). The samples
were incubated for 30 min at room temperature. Subsequently, 5 μL
of donor beads (5×) were added to samples under low light conditions
and incubated for 30 min at room temperature. For all experiments,
samples were prepared in triplicate and the assay was repeated 2 times.
The AlphaLISA signal was detected with the Tecan Spark multimode microplate
reader using the following settings: mode: AlphaLISA, excitation time:
130 ms, and integration time: 300 ms. In α screen experiments,
the FKBP-rapamycin binding (FRB) protein was used as a negative control.
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