Surface exposure of Calreticulin (CRT) and Mannose-6 Phosphate receptor (CD220) was evaluated by staining with monoclonal antibodies specific for CRT (unconjugated, specific for both mouse and human, Abcam ab22683, and PE-conjugated, specific for human only, Abcam, ab83220) and CD220 (Thermo Schientific, MA1-10148) versus isotype controls. Staining for CRT was performed as previously described64 (link). Cell viability/membrane integrity probe was used to discriminate/gate between live and dead cells (Life Technologies, L23105).
Ab22683
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab22683 is a primary antibody that specifically recognizes the human Myc proto-oncogene protein. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
L23105, by Thermo Fisher Scientific (1 mentions)
Ab109522, by Abcam (1 mentions)
Donkey anti goat igg h l, by Jackson ImmunoResearch (1 mentions)
Rabbit anti tom20 sc 11415, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 donkey anti rabbit igg a21206, by Thermo Fisher Scientific (1 mentions)
Ab13506, by Abcam (1 mentions)
Ab92573, by Abcam (1 mentions)
Mab1273, by Merck Group (1 mentions)
Fluorophore coupled secondary antibodies, by Abcam (1 mentions)
Prolong diamond with dapi, by Thermo Fisher Scientific (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Storm optical scanner, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Luminata crescendo western hrp substrate, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti calreticulin antibody, by Abcam (1 mentions)
Fitc conjugated annexin 5, by Immunostep (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Elisa, by Solarbio (1 mentions)
11 protocols using ab22683
1
Evaluating Cell Surface Markers: CRT and CD220
2
Immunoblotting and Immunofluorescence Protocols
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Rabbit anti-ASAH1 was from Aviva Systems Biology (Cedarlane, Burlington, ON, Canada (OAPB00726; WB 1:750). Mouse monoclonal anti-SPHK1 (SC-365401; IF 1:200), goat anti-SPHK2 (SC-22704; IF 1:200, WB 1:500), rabbit anti-ZO-1 (SC-10804; IF 1:200), and rabbit anti-TOM20 (SC-11415; IF 1:200) were purchased from Santa Cruz Biotechnology (Mississauga, ON, Canada). Rabbit monoclonal anti-SPHK1 (ab109522; WB 1:1000) and mouse monoclonal anti-calreticulin (ab22683; IF 1:300) were from Abcam (Cedarlane, Burlington, ON, Canada). Secondary antibodies used were goat anti-rabbit IgG-HRP (sc-2054; WB 1:2000), goat anti-mouse IgG-HRP (sc-2005; WB 1:2000), and donkey anti-goat IgG (H+L) (WB 1:2000) from Jackson Laboratory, Bar Harbor, Maine, USA. For IF experiments, Alexa Fluor 488 donkey anti-rabbit IgG (A21206) and Alexa Fluor 594 donkey anti-mouse IgG (A21203) were obtained from ThermoFisher Scientific (Mississauga, ON, Canada).
3
HERG Protein Complex Identification
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293 cells expressing HERG in 6-well cell culture plates were harvested at 48 h after transient transfection as aforementioned. Subsequently, the IP/CoIP kit (cat. no. abs955; Absin (Shanghai) Biotechnology Co., Ltd.) was used. After centrifugation at 14,000 × g for 10 min at 4°C, the supernatant was the cell division product. Subsequently, beads (5 µl Protein A and 5 µl Protein G) were added to 500 µl (containing 200-1,000 µg total protein) cell lysate. CNX-HERG, CRT-HERG and ERP57-HERG complexes were immunoprecipitated by incubation with 2 µg antibody against CNX (cat. no. ab92573; Abcam), CRT (cat. no. ab22683; Abcam) and ERP57 (cat. no. ab13506; Abcam), respectively, at 4°C overnight. Furthermore, 5 µl Protein A and 5 µl Protein G were added and mixed gently at 4°C for 1-3 h, then the precipitate was washed with 0.5 ml 1X Wash buffer (from the IP/CoIP kit), centrifuged at 12,000 × g for 1 min at 4°C, and the precipitate was retained. Subsequently, 20-40 µl 1X SDS was added to the precipitate, and the sample was heated to 100°C for 5 min. This was followed by analysis by western blotting and band densities were quantitated using ImageJ.
4
Immunofluorescence Assay of THEM6 and CALR
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Cells seeded on glass coverslips were fixed in ice‐cold Methanol/Acetone buffer, washed, blocked in 5% BSA and probed with primary antibodies overnight (anti‐THEM6, ab121743, anti‐CALR, ab22683, Abcam, Cambridge, UK; anti‐mitochondria, MAB1273, Merck Millipore, Burlington, MA, USA). The next day, coverslips were washed and incubated with fluorophore‐coupled secondary antibodies (Abcam, Cambridge, UK). Coverslips were mounted using Diamond Prolong with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Pictures were taken on a Nikon A1R confocal microscope (Nikon Instruments Europe B.V., Amsterdam, The Netherlands).
5
Western Blot Analysis of Cellular Proteins
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Proteins (45μg) in cell extracts and conditioned media were separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% nonfat dry milk for 2h at room temperature, and incubated with primary antibodies against GSN (1:1500, Proteintech, 11644-2-AP), PPIA (1:2000, Proteintech, 10720-1-AP), TXN (1:200, Santa Cruz, sc-20146), ADAMTSL4 (1:500, Proteintech, 11644-2-AP), CALR (1:1000, Abcam, ab22683) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10000, Abcam) for 1 h at room temperature. The signal was visualized with Luminata Crescendo Western HRP Substrate (Millipore) and quantitated by densitometry using Image Quant image analysis system (Storm Optical Scanner, Molecular Dynamics, Sunnyvale, CA).
6
Cell Death Profiling by Flow Cytometry
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For cell death profiling, cells were treated with the indicated OR141 concentrations, trypsinized and washed with PBS. Cells were consecutively incubated with fluorescein isothiocyanate (FITC)-conjugated Annexin V (Immunostep, Salamanca, Spain, ANXVF-200T) and 1 μg/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA), according to manufacturer’s instructions. After 15 min incubation at room temperature in the dark, cells were analyzed by flow cytometry on FACSCanto II (BD Pharm) with a gating strategy, excluding debris and doublet cells. For calreticulin translocation, cells were treated as described above and gently scraped off the plate after 6 h. After staining with an anti-calreticulin antibody (Abcam, Cambridge, UK, ab22683), a secondary goat anti-mouse APC-coupled antibody was added for 15 min at RT. Cells were then counterstained with PI and analyzed on FACSCanto II.
7
Cell Surface CRT and HMGB1 Release Assay
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The analysis of cell surface CRT was performed by flow cytometry. The cells were collected and incubated with primary mouse anti-CRT (Abcam, ab22683) for 30 min at 4°C. Then, cells were washed and stained with the secondary antibody. The live cells were gated as DAPI-. HMGB1 in culture supernatants was measured by ELISA (Solarbio Life Science, China). ATP levels in culture supernatants were measured by CellTiter-Glo® Luminescent Cell Viability Assay.
8
Protein Expression Analysis in Neonatal Rat Cardiomyocytes
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Whole-cell proteins were isolated from neonatal rat ventricular myocytes. Samples
containing 100 μg of protein in 10 µL of loading buffer (Beyotime Biotechnology, Shanghai,
China) were loaded and separated by electrophoresis on 8% sodium-dodecyl sulfate
polyacrylamide gels. Then, proteins in the gels were transferred to polyvinylidene
fluoride membranes and blocked for 1 hour using 5% nonfat milk, followed by incubation
with primary antibodies to calreticulin (ab22683, Abcam, Cambridge, UK, observed band size
65 kDa) and CaV1.3 (ab84811, Abcam, observed band size 245 kDa) overnight at
4°C on a shaker. The membranes were washed three times with 0.05% phosphate buffered
saline plus 0.05% Tween and incubated with corresponding fluorescent secondary antibodies
(926-32211 and 926-32210, LI-COR Biosciences, Lincoln, NE, USA) for 1 hour in the dark at
room temperature. Finally, the bands on the membranes were detected with the Odyssey
instrument (LI-COR Biosciences), and Odyssey software v1.2 was used to analyze and
quantify the bands. The intensities of proteins were normalized to the respective β-actin
intensity of each gel.
containing 100 μg of protein in 10 µL of loading buffer (Beyotime Biotechnology, Shanghai,
China) were loaded and separated by electrophoresis on 8% sodium-dodecyl sulfate
polyacrylamide gels. Then, proteins in the gels were transferred to polyvinylidene
fluoride membranes and blocked for 1 hour using 5% nonfat milk, followed by incubation
with primary antibodies to calreticulin (ab22683, Abcam, Cambridge, UK, observed band size
65 kDa) and CaV1.3 (ab84811, Abcam, observed band size 245 kDa) overnight at
4°C on a shaker. The membranes were washed three times with 0.05% phosphate buffered
saline plus 0.05% Tween and incubated with corresponding fluorescent secondary antibodies
(926-32211 and 926-32210, LI-COR Biosciences, Lincoln, NE, USA) for 1 hour in the dark at
room temperature. Finally, the bands on the membranes were detected with the Odyssey
instrument (LI-COR Biosciences), and Odyssey software v1.2 was used to analyze and
quantify the bands. The intensities of proteins were normalized to the respective β-actin
intensity of each gel.
9
Co-Immunoprecipitation of CRT and Fas
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For co-IP, 500 μL PBS with protease inhibitor (1 : 100, Sigma-Aldrich, St. Louis, MO, USA) was used to dilute 500 μg protein. The solution was preincubated with anti-CRT antibody (12.5 μg/mL, Abcam, ab22683) or anti-Fas antibody (1 μg/mL, Santa Cruz Biotechnology Inc., sc-21730, Santa Cruz, CA, USA) at 4°C overnight on a rotating shaker. Additional rotation for 2 h was continued after addition of 20 μL protein A/G-Sepharose bead slurry (Millipore, Billerica, MA, USA) to the mixture. Ice-cold cell lysis buffer was used to wash the slurry three times. Proteins were eluted with SDS sample buffer and boiled for 5 min. Supernatant was subjected to SDS-PAGE and Western blotting using anti-FasL antibody (1 : 200, Santa Cruz Biotechnology, Inc., sc-834).
10
GC Cell Immunogenicity Assay
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Human (MKN7, MKN45, and KATOIII) and mouse (T3-2D) GC cells were infected with OBP-702 (0, 10, or 50 MOI) for 24 h (n = 5), and levels of extracellular ATP in supernatants were measured using an ENLITEN ATP assay (Promega, Madison, WI, USA), according to the protocols from the manufacturer. HMGB-1 and calreticulin assays were performed as follows. Cells were incubated with Fc block, followed by CD16/32 antibodies (Thermo Fisher Scientific, Waltham, MA, USA). Cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) to detect live cells. Flow Cytometry was performed on a FACSArray (BD Biosciences, San Jose, CA, USA), and analyzed by the FlowJo program (BD Biosciences, Franklin Lakes, NJ, USA). The antibodies used for flow cytometry were mouse anti-HMGB-1 Ab (MA5-16263, Invitrogen, Waltham, MA, USA) and mouse anti-Calreticulin Ab (ab22683, Abcam plc).
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