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Itlc sa

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The ITLC-SA is a type of thin-layer chromatography (TLC) plate used for the separation and analysis of chemical compounds. It is composed of a silica gel-based stationary phase coated on an aluminum backing. The ITLC-SA plate provides a stable and uniform surface for the separation of analytes based on their relative affinities to the stationary phase and mobile phase during chromatographic separation.

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Lab products found in correlation

Crc 25 dose calibrator, by Mirion Technologies (4 mentions) Amersham typhoon bioimager, by GE Healthcare (4 mentions) Imagequant software, by GE Healthcare (4 mentions) Wizard 2480 gamma counter, by PerkinElmer (3 mentions) Microflex lrf, by Bruker (3 mentions) Nanodrop one microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions) Nanodrop onec microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions) Ph indicator paper, by Merck Group (1 mentions) Ar2000 tlc scanner, by Eckert & Ziegler (1 mentions) Ultraflextreme instrument, by Bruker (1 mentions) Avance 3 hd spectrometer, by Bruker (1 mentions) Ar 2000 radio tlc plate reader, by Bioscan (1 mentions) Inveon animal pet scanner, by Siemens (1 mentions) Automatic wizard2 link γ counter, by PerkinElmer (1 mentions) Winscan radio tlc software, by Bioscan (1 mentions)

7 protocols using itlc sa

1

Radiochemical Synthesis and Stability

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All reagents were purchased from Thermo
Fisher Scientific unless otherwise stated and used without further
purification. Water was deionized using a Select Fusion ultrapure
water deionization system (Suez) and had a resistance of >18.2
MΩ
cm–1 at 25 °C. Protein concentration measurements
were obtained using a NanoDrop One Microvolume UV–vis spectrophotometer
(NanoDrop Technologies, Inc.). Mass spectrometry measurements were
performed using a Thermo RSLC coupled to Bruker maXis. Radioactivity
measurements were obtained using a CRC-25 Dose Calibrator (Capintec,
Inc.) or a Wizard 2480 Gamma Counter (PerkinElmer). RIC synthesis
and serum stability studies were monitored by instant thin-layer chromatography
using glass microfiber chromatography paper (iTLC-SA, Agilent). Radio-iTLC
strips were measured by autoradiography (Amersham Typhoon Bioimager,
GE) and analyzed using ImageQuant software (GE Healthcare). All experiments
were performed in accordance with the United Kingdom Human Tissue
Act (2004) regulations. Appropriate informed consent for the use of
human tumor specimen slides and paraffin embedded blocks was obtained
and approved by the Newcastle and North Tyneside 1 Research Ethics
Committee (REC reference number: 17/NE/0361).
Pringle T.A., Chan C.D., Luli S., Blair H.J., Rankin K.S, & Knight J.C. (2022). Synthesis and In Vivo Evaluation of a Site-specifically Labeled Radioimmunoconjugate for Dual-Modal (PET/NIRF) Imaging of MT1-MMP in Sarcomas. Bioconjugate Chemistry, 33(8), 1564-1573.
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2

Radioimmunoconjugate Synthesis and Stability

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All reagents were purchased from Fisher Scientific unless otherwise stated and used without further purification. Water was deionized using a Select Fusion ultrapure water deionisation unit (Suez) and had a resistance of >18.2 MΩ cm−1 at 25 °C. Protein concentration measurements were obtained using a NanoDrop OneC Microvolume UV-vis Spectrophotometer (NanoDrop Technologies, Inc.). MALDI-TOF mass spectrometry measurements were taken on a Bruker Microflex LRF. Radioactivity measurements were obtained using a CRC-25 Dose Calibrator (Capintec, Inc.) or a Wizard 2480 Gamma Counter (PerkinElmer). Radioimmunoconjugate synthesis and serum stability studies were monitored by instant thin-layer chromatography using glass microfiber chromatography paper (iTLC-SA, Agilent). Radio-iTLC strips were measured by autoradiography (Amersham Typhoon Bioimager, GE) and analysed using ImageQuant software (GE Healthcare). pH measurements were determined using pH indicator paper (Merck Millipore) or a pH Spear electrode (Eutech Instruments).
Pringle T.A., Coleman O., Kawamura A, & Knight J.C. (2022). The influence of degree of labelling upon cellular internalisation of antibody-cell penetrating peptide conjugates. RSC Advances, 12(43), 27716-27722.
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3

Radiolabeling of Pb-203-Ligand Complexes

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Stock solutions of
the ligands (10–3 M) were prepared in ultrapure
deionized H2O and diluted appropriately to give a serial
dilution series (10–4–10–6 M). Concentration-dependent radiolabeling was performed by the addition
of [203Pb]Pb2+ (10 μL, 124 kBq) to a solution
containing the ligand (10 μL, 10–3–10–6 M) diluted in NH4OAc buffer (80 μL,
1 M, pH = 7). Water replaced the ligands in the negative control.
All of the radiolabeling for the cyclen-based derivatives was performed
at room temperature and monitored at 5 min and 1 h time points whereas
heating at T = 80 °C was also employed for TACD3S,
TRI4S, and TE4S. All radiolabeling reactions were repeated at least
in triplicate.
Radiochemical incorporation (RCI) was determined
via instant thin-layer chromatography (iTLC) with silicic acid (SA)-impregnated
paper TLC plates (iTLC-SA, Agilent Technologies, USA). Ethylenediamine
tetraacetic acid (EDTA, 50 mM, pH = 5.5) was used as the eluent. Under
these conditions, free [203Pb]Pb2+ migrates
with the solvent front (Rf = 1) while
the [203Pb]Pb2+ complexes remain at the baseline
(Rf = 0). The iTLC plates were analyzed
on an Eckert & Ziegler AR-2000 TLC scanner, and all the data were
processed with Eckert & Ziegler WinScan software. Representative
TLC radiochromatograms are presented in Figure S20.
Tosato M., Randhawa P., Lazzari L., McNeil B.L., Dalla Tiezza M., Zanoni G., Mancin F., Orian L., Ramogida C.F, & Di Marco V. (2024). Tuning the Softness of the Pendant Arms and the Polyazamacrocyclic Backbone to Chelate the 203Pb/212Pb Theranostic Pair. Inorganic Chemistry, 63(4), 1745-1758.
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4

Radiolabeled Conjugate Characterization

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All chemicals, unless otherwise noted, were purchased from Sigma-Aldrich, and used as received without further purification. Ultrapure water produced by a PURELAB Ultra system from ELGA was used throughout (18.2 MΩ cm). NMR spectra were acquired on a Bruker Avance III HD spectrometer operating at 600 MHz. The conjugates were purified by FPLC on an Äkta Pure 25 M chromatography system (GE Healthcare Life Sciences). MALDI-TOF mass spectra were acquired on an ultrafleXtreme instrument (Bruker Daltonics). Size-exclusion chromatography (SEC-HPLC) analyses were performed on a JASCO HPLC system LC-2000 analytical series equipped with a Superdex 200 5/150 GL column. Instant thin-layer chromatography (iTLC) was performed using sheets impregnated with salicylic acid (iTLC-SA; Agilent) eluted with 0.1 M EDTA, pH 5.0, and analysed on an AR-2000 radio-TLC plate reader (Bioscan Inc.). PET/CT scans were acquired on an Inveon animal PET scanner (Siemens Preclinical Solutions).
Raavé R., Sandker G., Adumeau P., Jacobsen C.B., Mangin F., Meyer M., Moreau M., Bernhard C., Da Costa L., Dubois A., Goncalves V., Gustafsson M., Rijpkema M., Boerman O., Chambron J.C., Heskamp S, & Denat F. (2019). Direct comparison of the in vitro and in vivo stability of DFO, DFO* and DFOcyclo* for 89Zr-immunoPET. European Journal of Nuclear Medicine and Molecular Imaging, 46(9), 1966-1977.
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5

Chelator-free Radiolabeling of cRGDY-PEG-C' Dots

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For a typical chelator-free 89Zr labeling of cRGDY-PEG-C′ dots, 4 nmol of cRGDY-PEG-C′ dots was mixed with 1 mCi of 89Zr-oxalate in HEPES buffer (pH 8) at 75 °C. The radiolabeling yield of cRGDY-PEG-C′ dots at 1, 30, 60, 120, and 240 min was monitored using salicylic acid impregnated instant thin-layer chromatography paper (ITLCSA) (Agilent Technologies), and analyzed either on a Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-TLC software (Bioscan Inc., Washington, DC) or an Automatic Wizard2 (link)γ-Counter (PerkinElmer). After incubation, 5 μL aliquots were withdrawn and mixed with 50 μL of EDTA (50 mM, pH 5–6) before analyzing by ITLC using EDTA (50 mM, pH 5–6) as a mobile phase solvent. Free 89Zr formed an instantaneous complex with EDTA and eluted with the solvent front, while 89Zr-labeled cRGDY-PEG-C′ dots remained at the origin. For more accurate quantification, strips were cut in half, and γ-ray emissions at 909 keV were counted on a calibrated γ-counter (PerkinElmer) using a dynamic energy window of 800–1000 keV. Similar procedures were introduced when studying the pH-, concentration-, and temperature-dependent chelator-free labeling of cRGDY-PEG-C′ dots. The specific activity of chelator-free 89Zr-labeled cRGDY-PEG-C′ dots was found to be in the range of 100–500 Ci/mmol.
Chen F., Ma K., Zhang L., Madajewski B., Zanzonico P., Sequeira S., Gonen M., Wiesner U, & Bradbury M.S. (2017). Target-or-Clear Zirconium-89 Labeled Silica Nanoparticles for Enhanced Cancer-Directed Uptake in Melanoma: A Comparison of Radiolabeling Strategies. Chemistry of materials : a publication of the American Chemical Society, 29(19), 8269-8281.
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6

Radioimmunoconjugate Synthesis and Evaluation

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All reagents were purchased from Thermo Fisher Scientific unless otherwise stated and used without further purification. Water was deionized using a Select Fusion ultrapure water deionisation system (Suez) and had a resistance of > 18.2 MΩ cm–1 at 25 °C. Protein concentration measurements were obtained using a NanoDrop One Microvolume UV–Vis Spectrophotometer (NanoDrop Technologies, Inc.). MALDI-TOF mass spectrometry measurements were taken on a Bruker Microflex LRF. Radioactivity measurements were obtained using a CRC-25 Dose Calibrator (Capintec, Inc.) or a Wizard 2480 Gamma Counter (PerkinElmer). Radioimmunoconjugate synthesis and serum stability studies were monitored by instant thin-layer chromatography using glass microfiber chromatography paper (iTLC-SA, Agilent). Radio-iTLC strips were measured by autoradiography (Amersham Typhoon Bioimager, GE) and analysed using ImageQuant software (GE Healthcare).
Pringle T.A., Ramon-Gil E., Leslie J., Oakley F., Wright M.C., Knight J.C, & Luli S. (2024). Synthesis and preclinical evaluation of a 89Zr-labelled human single chain antibody for non-invasive detection of hepatic myofibroblasts in acute liver injury. Scientific Reports, 14, 633.
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7

Radiolabeling Immunoconjugate Protocol

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All materials were obtained from Fisher
Scientific unless otherwise specified and used without any further
purification. Deionized water was obtained using a Select Fusion ultrapure
water deionization system (Suez) with a resistance of >18.2 MΩ/cm
at 25 °C. Absorbance measurements were obtained using a NanoDrop
OneC Microvolume UV–vis spectrophotometer (NanoDrop
Technologies, Inc.). MALDI-TOF mass spectrometry analysis was performed
by using a Bruker Microflex LRF. Radioactivity measurements were obtained
by using a CRC-25 dose calibrator (Capintec, Inc.). Radiolabeling
of immunoconjugates was verified by instant thin-layer chromatography
(iTLC) using glass microfiber chromatography paper (iTLC-SA, Agilent).
Autoradiography of radio-iTLC strips was imaged using an Amersham
Typhoon bioimager (GE) and analyzed using ImageQuant software (GE
Healthcare).
Gristwood K., Luli S., Rankin K.S, & Knight J.C. (2023). Synthesis and In Vitro Evaluation of a HER2-Specific ImmunoSCIFI Probe. ACS Omega, 8(50), 47905-47912.
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