The dissociation constant for the LRP1–SAA1 interaction was determined by microscale thermophoresis (48 (link)). Purified SAA1 was labeled with AlexaFluor 594 using the Microscale Protein Labeling Kit (Invitrogen). Labeled SAA1 was incubated with retinol (or DMSO) at a 3:1 molar ratio for 2 hours at 4°C in MST buffer (150 mM NaCl, 5 mM CaCl2, 0.05% (v/v) Tween 20, and 20 mM HEPES, pH 6.5). Next, a 16-fold titration series of purified LRP1 ectodomain (0 to 1.75 μM) diluted 1:1 with the MST buffer was mixed with SAA1 at 50 nM. The mixtures containing diluted LRP1 and SAA1 were incubated for 30 min at room temperature in the dark to enable binding. Each titration sample was then loaded into a premium capillary (NanoTemper) and analyzed on a Monolith NT.115 Pico (NanoTemper). LRP1-dependent fluorescence enhancement was observed, and absolute fluorescence was used to calculate the Kd. The specificity of the binding event was carefully validated by the SDS-denaturation test according to the manufacturer’s protocol. Binding isotherms were deduced by using PALMIST software (49 (link)) and the graph was created in GUSSI (50 (link)).
Microscale protein labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Microscale Protein Labeling Kit is a set of reagents and protocols designed for the covalent labeling of proteins on a microscale. The kit enables the attachment of various fluorescent dyes or other labels to proteins for downstream detection and analysis applications.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Dm6000b, by Leica camera (2 mentions)
Efluor 780 fixable viability live dead dye, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Lightsheet z 1 microscope, by Zeiss (2 mentions)
Monolith nt 115 pico, by NanoTemper (1 mentions)
Novoexpress software, by Agilent Technologies (1 mentions)
Apc anti mouse cd11c clone hl3, by BD (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Clone l243, by BD (1 mentions)
Mouse igg2a, by BD (1 mentions)
Eclipse e1000, by Nikon (1 mentions)
Anti cd44, by Cell Signaling Technology (1 mentions)
Anti cxcr4, by Abcam (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Irdye800cw microscale kit, by LI COR (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Limulus amebocyte lysate, by Thermo Fisher Scientific (1 mentions)
Thiazole orange, by Merck Group (1 mentions)
22 protocols using microscale protein labeling kit
1
Microscale Thermophoresis Measurement of LRP1-SAA1 Interaction
2
Investigating BMDC Activation by CpG/DP7 Complex
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NY-ESO-1 was labeled with Alexa Fluor® 594 with the Microscale Protein Labeling Kit (Invitrogen, Carlsbad, CA, USA). The labeled NY-ESO-1 was incubated with DP7 (40 µg/ml), CpG (20 µg/ml), or the CpG/DP7 combination for 10 min and then incubated with a culture of BMDCs for 1 h. Cell membranes tagged with green fluorescent dye. BMDCs were washed three times with PBS, fixed in 4% paraformaldehyde, and stained with DAPI. Finally, the BMDCs were observed under a confocal laser scanning microscope.
To study the effect of the CpG/DP7 complex on BMDCs, BMDCs were incubated with DP7 (40 µg/ml), CpG (20 µg/ml) or the CpG/DP7 combination for 16 h, stained with APC-anti-Mouse CD11c (Clone HL3; BD Biosciences Cat# 550261), FITC-anti-mouse-CD40 (Clone 3/23; BD Biosciences Cat# 561845), PerCP-Cy5.5-anti-mouse-CD80(clone 16-10A1; BD Biosciences Cat# 560526) or FITC-anti-mouse-CD86 (clone GL1; BD Biosciences Cat# 561962). BMDCs were gated as CD11c+ and analyzed for CD40, CD80, CD86 expression by NovoExpress software (ACEA Biosciences, Inc.).
To study the effect of the CpG/DP7 complex on BMDCs, BMDCs were incubated with DP7 (40 µg/ml), CpG (20 µg/ml) or the CpG/DP7 combination for 16 h, stained with APC-anti-Mouse CD11c (Clone HL3; BD Biosciences Cat# 550261), FITC-anti-mouse-CD40 (Clone 3/23; BD Biosciences Cat# 561845), PerCP-Cy5.5-anti-mouse-CD80(clone 16-10A1; BD Biosciences Cat# 560526) or FITC-anti-mouse-CD86 (clone GL1; BD Biosciences Cat# 561962). BMDCs were gated as CD11c+ and analyzed for CD40, CD80, CD86 expression by NovoExpress software (ACEA Biosciences, Inc.).
3
Antibody Purification and Labeling
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For the studies using antibodies directly labeled with fluorescent dye, Protein A agarose columns (Invitrogen, Carlsbad, CA, USA) were used to purify the IgG fraction of antibodies prior to labeling according to manufacturers' instructions. The IgG concentration of each eluate was determined using a spectrophotometer. IgG fractions of antibodies to big LEN and SAAS were labeled with Alexa 488 using the Microscale Protein labeling kit (Invitrogen, Carlsbad,CA) according to the manufacturer's instructions.
4
Fluorescent Labeling of anti-mTF scFv and IgG
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Each of the anti-mTF scFv and IgG was conjugated with Alexa 647 using the Microscale Protein Labeling kit (Invitrogen) and Alexa Flour 647 protein labeling kit (Invitrogen), according to the manufacturer's instructions. The concentration of protein and fluorescence were determined by measuring the absorbance at 280 and 650 nm, respectively.
5
Labeling Immune Cell Antibodies
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Mouse anti-human HLA-DR antibody that targets APCs (BD, clone L243) and its isotype control (mouse IgG2a (BD)) were labeled with AlexaFluor647 using the Microscale Protein Labeling Kit (Invitrogen). Antibody and fluorophore concentrations were measured after coupling by nanodrop spectrophotometry.
6
Internalization of Recombinant MIF Protein
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Recombinant human MIF protein (rMIF, Cell Guidance Systems, Cambridge, UK) was conjugated with the AlexaFlour 546/488 dye with the Microscale Protein Labeling Kit (A30006; Invitrogen). PTC cell lines were seeded at 12.5!10 4 cells/cm 2 and cultured for 18 h. The medium was removed, and cells were washed twice with PBS to remove endogenous MIF. Alexa 546 MIF or Alexa 488 MIF (pre-treated overnight with 4-IPP or untreated) was added directly to fresh culture medium to achieve a final concentration of 400 ng/ml, and cells were cultured at 37 8C. After 0, 30, or 60 min, cells were washed, fixed in 4% paraformaldehyde, and stained with anti-CD74 (ImmunoTools, Friesoythe, Deutschland), anti-CD44 (Cell Signaling Technology, Inc., Boston, MA, USA), or anti-CXCR4 (Abcam, Inc.). DAPI stain (Biostatus Limited) was used as a nuclear marker. Slides were imaged with immunofluorescence microscopy (Eclipse E1000; Nikon Instruments, Inc.).
7
Fluorescent Labeling of DF Proteins
8
Fluorescent Labeling of DF Proteins
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For DF phase separation experiments, DF1, DF2 and DF3 proteins were fluorescently labeled using Alexa Fluor (488, 594 and 647, respectively) Microscale Protein Labeling kit according to the manufacturer’s instruction (A30006, A30008, A3009, Thermo Fischer Scientific). Briefly, DF proteins were diluted at 1 mg/mL in PBS and mixed with 100 mM sodium bicarbonate. The reaction was incubated for 15 min at room temperature and fluorescently labeled proteins were purified from the unreacted dye substrate by column purification using Micro Bio-Spin Columns with P-30 gel. The labeled protein was eluted in 20 mM HEPES pH 7.4, 300 mM KCl, 6 mM MgCl2, 0.02% NP-40 and buffer exchange was performed in two successive rounds using Amicon 0.5 mL Ultracel centrifugal filter columns. Protein labeling was performed the day of each experiment.
9
Visualizing Amelogenin Protein Uptake
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20kDA recombinant amelogenin (rAMG 20kD) was synthesized and purified as previously described44 (link). The purified rAMG 20 kD was labeled with Alexa Fluor 594, using a Microscale Protein Labeling Kit (Thermo Fisher Scientific, MA) (A30008).
Control and KO cell clones were grown to 70% confluence at equivalent cell densities on 35 mm glass bottom dishes No. 0 (MatTek) (P35GC-0-14-C/H), then starved in serum-free media for 24 h to facilitate endocytosis. Following starvation, 1 µL of 1 mg/mL labeled rAMG 20kD was added to media for 30 min. A solution of Hoescht DAPI stain (Invitrogen, H3570) and LysoTracker Green DND-26 (Life Technologies, CA, L7526) in 1× PBS/FBS were incubated with cells for 5 min. All cell groups were rinsed 3 times, and imaged under confocal microscopy for 45, 60, 90, and 135 min after rAMG 20kD was added.
Control and KO cell clones were grown to 70% confluence at equivalent cell densities on 35 mm glass bottom dishes No. 0 (MatTek) (P35GC-0-14-C/H), then starved in serum-free media for 24 h to facilitate endocytosis. Following starvation, 1 µL of 1 mg/mL labeled rAMG 20kD was added to media for 30 min. A solution of Hoescht DAPI stain (Invitrogen, H3570) and LysoTracker Green DND-26 (Life Technologies, CA, L7526) in 1× PBS/FBS were incubated with cells for 5 min. All cell groups were rinsed 3 times, and imaged under confocal microscopy for 45, 60, 90, and 135 min after rAMG 20kD was added.
10
SARS-CoV-2 Spike Protein Characterization
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SARS-CoV-2 SPs (recombinant SARS-CoV-2 SP (S-RBD; cat# RP-87678, HEK293 cell expressed and binds ACE2) and ovalbumin Alexa Fluor-488 conjugate (Cat# 034781; molecular weight 45 kDa) were obtained from Life Technologies Corporation, Carlsbad CA, USA. The SP (molecular weight 36 kDa) was labeled with NIRF using a kit (IRDye800CW Microscale kit, Li-COR Biosciences, Nebraska, USA) and by following the manufacturer’s instructions. This conjugation forms a stable amide bond and has been used in clinical trials (Li-COR Biosciences). SP was labeled separately with Alexa Fluor 555 using a kit (Microscale protein labeling kit; ThermoFisher Scientific; Waltham, MA, USA) and by following the manufacturer’s instructions. In addition, both labels were purified using 3 kDa molecular weight cutoff ultrafiltration filter (Amicon Ultra Centrifugal Filter, Millipore). There was no detectable dye in the filtrate. Anti-SARS-CoV-2 SP antibody (SARS-CoV-2 Spike Protein Monoclonal Antibody (cat# MA5-36087; immunogen SARS-CoV-2 SP that interact with HeLa cell expressed SARS-CoV-2) and anti-ACE2 antibody (ACE2 Recombinant Rabbit Monoclonal Antibody (Cat# MA532307; interact with mouse ACE2) were obtained from Life Technologies Corporation.
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