The following primary rabbit polyclonal antibodies were used: anti‐PDIR, anti‐ERp57, anti‐P5, and anti‐GRP170 (all from ThermoFisher, cat. # PA3‐007, PA3‐009, PA3‐008, and PA5‐27655, respectively). In addition, a rabbit polyclonal antibody to the HA‐tag (Sigma, cat. #H6908) and a goat polyclonal antibody to reticulocalbin (Santa Cruz, cat. #sc‐109422) were used. Mouse monoclonal antibodies used were anti‐ATF6 (Abcam, cat. #ab122897), anti‐GAPDH (ThermoFisher, cat. #AM4300), anti‐V5 (Invitrogen, cat. #R960‐25), anti‐myc clone 4A6 (Merck, cat. #05‐724), anti‐HA (Sigma, cat. #H3663), and anti‐BiP (BD Biosciences, cat. #610979). A rabbit monoclonal anti‐HDAC2 was also used (Abcam, cat. #32117). The following secondary antibodies were used for Western blotting: goat anti‐mouse IRDye 800 (cat. #10751195), goat anti‐mouse IRDye 680 (cat. #A32729), goat anti‐rabbit IRDye 800 (cat. #13477187), and goat anti‐rabbit IRDye 680 (cat. #35568, all from ThermoFisher). Secondary antibodies for immunofluorescence were sheep anti‐rabbit‐FITC (Sigma, cat. #F7512) and donkey anti‐mouse‐Texas Red (Abcam, cat. #ab6818).
Anti bip
Manufactured by BD
Sourced in Canada, United Kingdom, United States
Anti-BiP is a laboratory reagent used to detect the presence of the immunoglobulin heavy chain binding protein (BiP) in cellular samples. BiP is a chaperone protein involved in the folding and assembly of proteins within the endoplasmic reticulum. Anti-BiP can be used in various biochemical and cell biology applications to assess the expression and localization of BiP in experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti v5, by Thermo Fisher Scientific (3 mentions)
Anti atf6, by Abcam (2 mentions)
Anti p5, by Thermo Fisher Scientific (2 mentions)
Pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Gradient sds page gel, by Bio-Rad (2 mentions)
Anti rab27, by Santa Cruz Biotechnology (2 mentions)
Anti cd63, by BD (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti cd81, by Santa Cruz Biotechnology (2 mentions)
Anti tsg101, by Abcam (2 mentions)
Sigma ge28 9068 38, by Merck Group (2 mentions)
Anti cd9, by Abcam (2 mentions)
Anti calreticulin, by Abcam (2 mentions)
Anti flag m1, by Merck Group (2 mentions)
Irdye 680 and 800 nm, by LI COR (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Ha tag, by Merck Group (1 mentions)
Anti myc clone 4a6, by Merck Group (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti bip
1
Antibody Characterization for ER Stress
2
Antibody and Plasmid Characterization Protocol
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The following antibodies were used: anti-BiP (BD Biosciences, Mississauga, Canada), anti-albumin, anti-transferrin, and anti-mouse actin (Sigma-Aldrich), anti-GAPDH (mAb 6C5; Millipore, Toronto, Canada), anti-human or hamster PrP (mAb 3F4; Cedarlane, Burlington, Canada), anti-mouse PrP (mAb Sha31; Bertin Pharma, Montigny le Bretonneux, France), anti-human MHC I (mAb W6/32; Ziegler et al., 1982 (link)), anti-mouse MHC I (mAb Y3; Jones et al., 1981 (link)), anti-PDI (AssayDesigns, Farmingdale, NY), anti-CD90 (Abcam, Cambridge, MA), anti-mouse FKBP10 (BD Biosciences), anti-human FKBP10 (Abcam) and anti-mouse LAMP1 (mAb 1D4B; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA).
pcDNA3.1 plasmids encoding wild-type hamster PrPC or hamster PrPC with its N-terminal signal sequence replaced with that of rat osteopontin or bovine prolactin were obtained from R. Hegde (MRC Laboratory of Molecular Biology, Cambridge, United Kingdom). ΔSP-PrP that encodes mouse PrP lacking its N-terminal signal sequence was generated by PCR from full-length mouse PrP cDNA in pcDNA3.1 using the following primers: forward primer, 5′-ACGGGATCCATGAAAAAGCGGCCAAAGCCTGGAG; and reverse primer, 5′-CGAGCGGCCGCTCATCCCACGATCAGGAAGATGA. The fragment was ligated into a new pcDNA3.1 plasmid using BamHI and NotI restriction sites.
pcDNA3.1 plasmids encoding wild-type hamster PrPC or hamster PrPC with its N-terminal signal sequence replaced with that of rat osteopontin or bovine prolactin were obtained from R. Hegde (MRC Laboratory of Molecular Biology, Cambridge, United Kingdom). ΔSP-PrP that encodes mouse PrP lacking its N-terminal signal sequence was generated by PCR from full-length mouse PrP cDNA in pcDNA3.1 using the following primers: forward primer, 5′-ACGGGATCCATGAAAAAGCGGCCAAAGCCTGGAG; and reverse primer, 5′-CGAGCGGCCGCTCATCCCACGATCAGGAAGATGA. The fragment was ligated into a new pcDNA3.1 plasmid using BamHI and NotI restriction sites.
3
Extracellular Vesicle Protein Analysis
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EV-containing fractions were lysed in 1X RIPA lysis buffer. Protein concentrations were determined by BCA protein assay kit (Thermo Fisher). Equivalent total protein amounts from BH and EVs were separated on 4 − 15% stain-free pre-cast SDS-PAGE gradient gels (Bio-Rad) under non-reducing conditions and transferred onto PVDF membranes (Sigma Aldrich). After 1 h blocking in 5% non-fat milk solution (Bio-Rad 170–6404) at room temperature, membranes were incubated with anti-CD63 (1:1000 dilution), anti-Bip (1:1000 dilution) (BD Biosciences 556019 and 610978, respectively), anti-CD81 (1:1000 dilution), anti-Rab27 (1:1000 dilution) (Santa Cruz Biotechnology sc23962, sc74586), anti-TSG101 (1:500 dilution), anti-CD9 (1:500 dilution), anti-Syntenin (1:500 dilution), anti-Calnexin (1:2000 dilution), or anti-GM130 (1:1000 dilution) (the last five antibodies were Abcam ab125011, ab92726, ab133267, ab22595, and ab76154) overnight at 4°C. The membrane was washed 3 times for 8 min in PBST while shaking, then incubated with HRP-conjugated secondary antibody (1:10000 dilution) (Santa Cruz Biotechnology sc-2357, sc-516102) at room temperature for 1 h. After washing again in PBST, the enzyme-linked antibody was detected by incubation with Pico chemiluminescent substrate (Thermo Fisher 34580) and recording on film (Millipore Sigma GE28-9068-38).
4
Quantification of UPR Pathway Proteins
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Cells were lysed in 1X RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5) plus 1% protease inhibitor cocktail, 1% PMSF (200 mM) and 1% sodium orthovanadate (Santa Cruz Biotechnology, USA). Lysates were clarified by centrifugation at 8000×g for 5 min at 4°C and equal amounts of protein were fractionated by SDS-PAGE and subsequently transferred onto nitrocellulose membrane, immunoblots were visualized using Supersignal® West Pico Chemiluminescent substrate (Thermo Scientific, Rockford, USA). Proteins were detected with anti-Glucose 6 Phosphate Dehydrogenase (Novus Biologicals, USA); anti-PERK (phospho T981) (#1055, Elabscience, Huston, USA); anti-PERK (#3667, Elabscience, Huston, USA); anti-IRE1 (phospho S724) (ab48187, Abcam, Cambridge, UK); anti-IRE1 (ab ab37073, Abcam, Cambridge, UK); Anti-eIF2α (phosphor S51) (9721, Cell Signaling, USA); anti-α-Tubulin Antibody (#2144 Cell Signalling Technology, UK); Anti-Calreticulin (ab2907, Abcam, Cambridge, UK); Anti-Calnexin (ab22595, Abcam, Cambridge, UK); Anti-BiP (BD610978, BD Biosciences, San Jose, CA); Anti-ATF4 (sc-200, Santa Cruz, Dallas, USA); Anti-Chop (sc7351, Santa Cruz, Dallas, USA); Anti-XBP1 (ab198999, Abcam, Cambridge, UK) and Anti-GAPDH (ab9485, Abcam, Cambridge, UK) were used for assessing loading.
5
ER Stress Protein Regulation Analysis
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The following chemicals were used: thapsigargin (MP Biomedicals, Santa Ana, CA), tunicamycin and
kifunensine (Calbiochem, Billerica, MA), puromycin (InvivoGen, San Diego, CA), and bafilomycin A1,
MG-132, and DTT (Sigma-Aldrich, St. Louis, MO). For Western blot analyses, the following antibodies
were used: anti-FLAG M1 and anti-tubulin (Sigma-Aldrich), anti–α1-antitrypsin (Dako,
Glostrup, Denmark), anti–14-3-3z and anti-SEL1L (Santa Cruz Biotechnology, Dallas, TX),
anti–S-tag and anti–OS-9 (Abcam, Cambridge, England), anti–GRP94 9G10 and
anti-KDEL (Enzo, Farmingdale, NY), anti-GRP94 (provided by Ineke Braakman, Utrecht University,
Utrecht, Netherlands), anti-HA (Covance, Princeton, NJ), anti-BiP (BD Biosciences, San Jose, CA),
and anti-PERK and anti-JNK (Cell Signaling Technologies, Danvers, MA). Secondary antibodies were
obtained from LI-COR (IRDye 680 and 800 nm, for use with the LI-COR Odyssey system [Lincoln, NE])
and Santa Cruz Biotechnology (horseradish peroxidase conjugated).
kifunensine (Calbiochem, Billerica, MA), puromycin (InvivoGen, San Diego, CA), and bafilomycin A1,
MG-132, and DTT (Sigma-Aldrich, St. Louis, MO). For Western blot analyses, the following antibodies
were used: anti-FLAG M1 and anti-tubulin (Sigma-Aldrich), anti–α1-antitrypsin (Dako,
Glostrup, Denmark), anti–14-3-3z and anti-SEL1L (Santa Cruz Biotechnology, Dallas, TX),
anti–S-tag and anti–OS-9 (Abcam, Cambridge, England), anti–GRP94 9G10 and
anti-KDEL (Enzo, Farmingdale, NY), anti-GRP94 (provided by Ineke Braakman, Utrecht University,
Utrecht, Netherlands), anti-HA (Covance, Princeton, NJ), anti-BiP (BD Biosciences, San Jose, CA),
and anti-PERK and anti-JNK (Cell Signaling Technologies, Danvers, MA). Secondary antibodies were
obtained from LI-COR (IRDye 680 and 800 nm, for use with the LI-COR Odyssey system [Lincoln, NE])
and Santa Cruz Biotechnology (horseradish peroxidase conjugated).
6
Immunoblotting of ER Stress Markers
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The following antibodies were used: anti‐TMX1 (SIGMA, HPA003085), anti‐BiP (BD Biosciences, San Jose, CA, USA, 610978), anti‐α‐tubulin (Wako, 017‐25031), anti‐PDI (Enzo Life Sciences, Farmingdale, NY, USA, SPA‐890), anti‐P5 (Thermo Fischer Scientific, PA3‐008), anti‐phospho‐eIF2α (Cell Signaling Technology, Danvers, MA, USA, 9721), and anti‐eIF2α (Cell Signaling Technology, 9722).
7
Protein Extraction and Western Blot Analysis
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24 h after transfection, cells were harvested and disrupted in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Nonidet P-40/0, 0.25% deoxycholate, 1 mM EDTA, and 50 mM Tris, pH 7.4) in the presence of complete protease inhibitor mixture (Sigma-Aldrich). 20 brains from larvae or 100 whole larvae for each genotype were used for protein extraction. Samples were rinsed in radioimmunoprecipitation assay buffer with complete protease inhibitor mixture (Roche) and 1 mM PMSF (Sigma-Aldrich), homogenized in a 1-ml glass/Teflon potter (Wheaton), and then spun at 7,000 g for 10 min. Supernatants were collected and boiled for 5 min in Laemmli buffer (4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol, and blue bromophenol). Extracted proteins were separated by 4–12% SDS-PAGE (NuPAGE; Invitrogen), transferred onto polyvinylidene difluoride (Bio-Rad Laboratories) membranes, and probed using the following antibodies: anti-Actin (1:2,000; EMD Millipore), anti-Myc (1:1,000 [BD] or 1:1,000 [Roche]), anti-V5 (1:1,000; Invitrogen), anti-Marf (1:500), anti-BiP/Grp78 (1:1,000; BD), and anti-BiP (1:500).
8
Glycosylation Monitoring in Eukaryotic Cells
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The following reagents were used: kifunensine and 4-methylumbelliferyl-6-sulfo-N-acetyl-β-d -glucosaminide (Calbiochem, Billerica, MA); bafilomycin A1, MG-132, dimethyl sulfoxide (DMSO), biotin, streptavidin, brefeldin A, proline, glutamic acid, TMAO, and trehalose (Sigma, St. Louis, MO); Z-VAD(OMe)-FMK (Abcam, Cambridge, England). For Western blot analyses, the following antibodies were used: anti-FLAG M1 (Sigma); anti–14-3-3 and anti-SEL1L (Santa Cruz, Dallas, TX); anti–OS-9, anti-PDIA6, anti-calreticulin, anti-HEXA (α), anti-HEXB (β), and anti-PDIA1 (Abcam); anti-GRP94 9G10 (Enzo, Farmingdale, NY); anti-BiP (BD Biosciences, San Jose, CA); anti-GRP170 (kind gift from Linda Hendershot, St. Jude Children’s Research Hospital, Memphis, TN); anti-HA (Cell Signaling Technologies, Danvers, MA); and anti-V5 (Invitrogen, Carlsbad, CA). Secondary antibodies were obtained from Li-Cor, Lincoln, NE (IRDye 680 and 800 nm, for use with the Li-Cor Odyssey system).
9
Comprehensive Protein Profiling Protocol
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The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874). All other antibodies were from Cell Signalling.
10
Extracellular Vesicle Characterization
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EV-containing fractions were lysed in 1X RIPA lysis buffer. Protein concentrations were determined by BCA protein assay kit (Thermo Fisher). Equivalent total protein amounts from BH and EVs were separated on 4-15% stain-free pre-cast SDS-PAGE gradient gels (Bio-Rad) under non-reducing conditions and transferred onto PVDF membranes (Sigma Aldrich). After 1 hour blocking in 5% non-fat milk solution (Bio-Rad 170-6404) at room temperature, membranes were incubated with anti-CD63 (1:1000 dilution), anti-Bip (1:1000 dilution) (BD Biosciences 556019 and 610978, respectively), anti-CD81(1:1000 dilution), anti-Rab27 (1:1000 dilution) (Santa Cruz Biotechnology sc23962, sc74586), anti-TSG101 (1:500 dilution), anti-CD9 (1:500 dilution), anti-Syntenin (1:500 dilution), anti-Calnexin (1:2000 dilution), or anti-GM130 (1:1000 dilution) (the last five antibodies were Abcam ab125011, ab92726, ab133267, ab22595, and ab76154) overnight at 4°C. The membrane was washed 3 times for 8 minutes in PBST while shaking, then incubated with HRP-conjugated secondary antibody (1:10000 dilution) (Santa Cruz Biotechnology sc-2357, sc-516102) at room temperature for 1 hour. After washing again in PBST, the enzymelinked antibody was detected by incubation with Pico chemiluminescent substrate (Thermo Fisher 34580) and recording on film (Millipore Sigma GE28-9068 -38).
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