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7 protocols using nb600 363

1

HA-tagged TALEN protein expression

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Western blot (WB) analyses were performed to determine the expression of the HA epitope of TALENs in HEK-293T cells using anti-HA antibody (NB600-363, Novus Biologicals). Human FANCA protein was also analyzed in immortalized FA-A MEFs and in gene-edited FA-A MEFs as previously described26 (link),85 (link).
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2

ZFN Protein Expression Analysis

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Protein detection from cell lysates and concentrated retroviral supernatants was performed as previously described34 (link) with minor adjustments. Briefly, U2OS-EGFP cells transfected with ZFN expression plasmids were harvested 48 h after transfection, while 293T producer cells were collected 60 h post-transfection. Cells were lysed in 100 ml of ice-cold RIPA buffer composed of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 0.5% sodium deoxycholate, 1% Nonidet P-40 and a cocktail of protease inhibitors (Complete Mini, Roche Applied Science). Samples were separated by SDS-polyacrylamide gel electrophoresis (12%) and transferred to polyvinylidene difluoride membranes. ZFN and EGFP expression was detected using antibodies directed against the HA tag (NB600-363; Novus Biologicals, Littleton, CO) and EGFP (MAB3580; Millipore, Billerica, MA), respectively. Control staining was performed using anti-α-actin (sc-81178, Santa Cruz Biotechnology) or anti-p30 antibodies (kindly provided by Sandra K. Ruscetti, NCI Frederick, MA, USA). Proteins were visualised after staining with HRP-conjugated mouse anti-rabbit, (Dianova) mouse anti-rat (Dianova), or donkey anti-goat (Santa Cruz) secondary antibody using the WestPico Chemiluminescence substrate (Thermo Scientific).
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3

SARS-CoV-2 Spike Protein and Host Receptor Analysis

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All protein samples were run under reduced conditions by dilution in 6× SDS containing dithiothreitol (DTT) and 5% beta-mercaptoethanol (Fisher Scientific; ICN19483425). The protein was subsequently incubated in a heating block at 95°C for 15 min, run on a 4 to 15% SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with phosphate-buffered saline blocking buffer (LI-COR; 927-700001) and then probed with the following antibodies. Antibodies against SARS-CoV2 (2B3E5 from Thomas Moran and GTX632604 from GeneTex), ACE2 (66699-1-Ig from Proteintech and Rb ab108252 from Abcam), VSV-G (A00199 from GenScript), VSV-M (EB0011 from Kerafast), anti-HA (NB600-363 from Novus), and CoX IV (926-42214 from LI-COR) were used. For secondary staining, membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti-mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. Alexa Fluor 647 was detected using the ChemiDoc MP imaging system (Bio-Rad). Relative ACE2 or TMPRSS2 abundance was calculated by first normalizing abundance relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression and then normalization to wild-type expression.
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4

SARS-CoV-2 Protein Immunoblot Analysis

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All protein samples were run under reduced conditions by dilution in 6X SDS containing dithiothreitol (DTT) and 5% beta-mercaptoethanol (Fisher Scientific; ICN19483425). The protein was subsequently incubated in a heating block at 95°C for 15mins, run on a 4–15% SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with phosphate-buffered saline blocking buffer (LI-COR, 927–700001), and then probed with the indicated antibodies. Antibodies against SARS-CoV2 (2B3E5 from Dr. Thomas Moran and GTX632604 from GeneTex), ACE2 (66699–1-Ig from Proteintech and Rb ab108252 from abcam), VSV-G (A00199 from Genescript), VSV-M (EB0011 from Kerafast), anti-HA (NB600–363 from Novus), and CoX IV (926–42214 from LI-COR) were used. For secondary staining, membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti-mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. Alexa Fluor 647 was detected using the ChemiDoc MP imaging system (Bio-Rad). Relative ACE2 or TMPRSS2 abundance was calculated by first normalizing abundance relative to GAPDH expression, then normalizing to wild type expression.
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5

Immunoprecipitation of Nipah Virus Matrix Protein

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293T cells in 6-wells were transfected with 1 ug of expression constructs or empty pcDNA3 vector for a total of 2 ug per well, using BioT transfection reagent (Bioland) according to manufacturer protocol. At 24 hours post-transfection, cells were lysed in cold lysis buffer (1% NP-40, 100 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM EDTA, 1X protease inhibitor cocktail (Roche)), clarified at 14k rpm for 5 minutes at 4°C, and incubated with 3 ug of rabbit anti-NiV-M [50 (link)] or rabbit anti-HA (Novus, NB600-363) at 4°C overnight. Following incubation with protein G agarose (Pierce) for 2 hours at 4°C, the bound protein was washed 4 times with cold wash buffer (same composition as lysis buffer but with 0.2% NP-40), then eluted in reducing Laemmli SDS sample buffer at 95°C.
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6

Analysis of PDGFRα Signaling Pathway

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An antibody against PDGFRα (AF 1602) was purchased from R&D Systems (Minneapolis, MN, USA). The antibodies recognizing phospho-PDGFRα (pY849, #3170), phospho-PLCγ1 (pTyr783, #2821), PLCγ1 (#2822), phospho-STAT3 (pTyr705, D3A7, #9145), STAT3 (79D7, #4904), phospho-Akt (pSer473, D9E, #4060), Akt (#9272S), phospho-ERK1/2 (pThr202/pThr204, #9101) and ERK1/2 (137F5, #4695) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SUMO1 (sc-5308) and ubiquitin (sc-8017) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The α-tubulin antibody (T6074) was from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies against HA (NB600-363) and the transferrin receptor (TfR) (NB100-92243) were from Novus Biologicals (Centennial, CO, USA). Antisera recognizing Alix (HP95) [42 ] and PDGFRβ (CTβ) [43 ] were prepared in house. The secondary antibodies used for immunoblotting included goat anti-mouse IgG (62-6520), goat anti-rabbit IgG (65-6120) and rabbit anti-goat IgG (81-1620), were from Invitrogen (Waltham, MA, USA). The inhibitors MG132 (C2211) and chloroquine (C6628) were from Sigma-Aldrich (St Louis, MO, USA). The inhibitor ginkgolic acid (345,887) was purchased from Merck KGaA and bortezomib (#2244) was from Cell Signaling Technology (Danvers, MA, USA).
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7

Antibody Characterization and Detection

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The following commercial antibodies were used: SFPQ-specific mouse monoclonal antibody [sc-374502, Santa Cruz Biotechnologies, Dallas, TX, USA; western blotting (WB) 1:500, immunocytochemistry (ICC) 1:100], anti-HA tag rabbit polyclonal antibody (NB600-363, Novus Biologicals, Centennial, CO, USA; WB 1:1000, ICC 1:500), pGA repeat rabbit polyclonal antibody (24492-1-AP, Proteintech, Rosemont, IL, USA), pGR repeat rabbit polyclonal antibody (23978-1-AP, Proteintech), pGP repeat rabbit polyclonal antibody (24494-1-AP, Proteintech), pAP repeat rabbit polyclonal antibody (24493-1-AP, Proteintech) and pPR repeat rabbit polyclonal antibody (23979-1-AP, Proteintech). All DPR antibodies were used at a 1:1000 dilution for dot blots, including GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech), anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 488 Conjugate) (4408, Cell Signaling Technology, Danvers, MA, USA; dilution 1:1000), StarBright Blue 520 Fluorescent Secondary Antibodies (nos. 12005866 and 12005869, Bio-Rad, Hercules, CA, USA; dilution 1:5000), StarBright Blue 700 Fluorescent Secondary Antibodies (nos. 12004158 and 12004161, Bio-Rad; dilution 1:5000) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-045, Jackson ImmunoResearch, West Grove, PA, USA; dilution 1:5000).
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