Protein samples were mixed with 4x LDS sample buffer (Invitrogen NP0007) and 10x sample reducing agent (Invitrogen NP0007) to a final concentration of 1×. Samples were loaded on 4–12% Bis-Tris gradient gel (Invitrogen 12-well, NP0322; 15-well, NP0323). The proteins were transferred to Immobilon-FL PVDF membrane (EMD Millipore, IPFL00010). The membrane was blocked with Odyssey blocking buffer (Li-COR, 927-40000) for 1 h at room temperature, followed by incubation with primary antibody in PBS overnight at 4 °C. The membrane was then washed with PBST (PBS, pH 7.4, and 0.1% Tween-20) three times and incubated with secondary antibody in PBS for another hour. After three washes with PBS, the membrane was scanned using an Odyssey imaging system (LI-COR) according to the manufacturer’s protocol. Quantification of western blots was carried out using the gel analysis function in ImageJ within the linear range of detection which is determined by using serial dilutions of a representative sample.
Np0007
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland, Italy
The NP0007 is a laboratory instrument designed for sample preparation and analysis. It features precise temperature control and advanced functionality to facilitate various experimental procedures. The device's core function is to provide a controlled environment for sample processing and analysis, enabling accurate and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Np0009, by Thermo Fisher Scientific (15 mentions)
Pierce ecl, by Thermo Fisher Scientific (12 mentions)
Np0004, by Thermo Fisher Scientific (8 mentions)
Np0005, by Thermo Fisher Scientific (7 mentions)
Imagequant tl, by Cytiva (6 mentions)
Np0336box, by Thermo Fisher Scientific (5 mentions)
Np0002, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (3 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (3 mentions)
Supersignal west femto ecl substrate, by Thermo Fisher Scientific (3 mentions)
Nxa931, by GE Healthcare (3 mentions)
Na934, by GE Healthcare (3 mentions)
β actin actb, by Merck Group (3 mentions)
Tuba4a tuba1a α tubulin, by Merck Group (3 mentions)
Vcl vinculin, by Merck Group (3 mentions)
Anti flag m2 magnetic bead, by Merck Group (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
A39255, by Thermo Fisher Scientific (2 mentions)
68 protocols using np0007
1
Western Blot Quantification Protocol
2
RBD-VLP Protein Desorption Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formulated RBD-VLP samples were centrifuged at 4000g for 10 min at 4°C. The supernatant with unbound protein was transferred to another tube without disturbing the pellet and prepared for SDS–polyacrylamide gel electrophoresis (SDS-PAGE) in 50 mM dithiothreitol (Thermo Fisher Scientific, A39255), 1× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, NP0007), and 20 mM iodoacetamide (Thermo Fisher Scientific, A39271), and heated at 95°C for 15 min. The alum-bound protein in the pellet was desorbed by resuspending the pellet in an elution buffer (0.4 M sodium phosphate, 50 mM dithiothreitol, 1× LDS sample buffer, and 20 mM iodoacetamide) and heating the samples at 95°C for 15 min. The desorbed samples were then centrifuged at 4000g for 10 min at 4°C to obtain desorbed protein in the supernatant for SDS-PAGE. RBD-SpyTag and HBsAg-SpyCatcher controls were prepared for SDS-PAGE similar to unbound protein sample. Approximately 0.1 μg of each sample was loaded on 4 to 12% bis-tris gel (Invitrogen, NP3022) run for 50 min at 150 V in 1× MES-SDS running buffer (Invitrogen, NP0002). Novex Sharp Pre-stained protein standard (Invitrogen, LC5800) was used as marker to estimate molecular weight of the proteins. The gel was stained using a Pierce Silver Stain kit (Thermo Fisher Scientific, 24612) as per manufacturer’s protocol and imaged using ProteinSimple FluorChem E imaging system.
3
RBD-VLP Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formulated RBD-VLP samples were centrifuged at 4000 g for 10 min at 4°C. The supernatant with unbound protein was transferred to another tube without disturbing the pellet and prepared for SDS-PAGE in 50 mM dithiothreitol (Thermo scientific, A39255), 1X LDS sample buffer (Invitrogen, NP0007), and 20 mM iodoacetamide (Thermo scientific, A39271), and heated at 95°C for 15 min. The alum-bound protein in the pellet was desorbed by resuspending the pellet in an elution buffer (0.4 M sodium phosphate, 50 mM dithiothreitol, 1X LDS sample buffer, and 20 mM iodoacetamide) and heating the samples at 95°C for 15 min. The desorbed samples were then centrifuged at 4000 g for 10 min at 4°C to obtain desorbed protein in the supernatant for SDS-PAGE. RBD-SpyTag and HBsAg-SpyCatcher controls were prepared for SDS-PAGE similar to unbound protein sample. Approximately 0.1 μg of each sample was loaded on 4–12% Bis-Tris gel (Invitrogen, NP3022) run for 50 min at 150 V in 1X MES-SDS running buffer (Invitrogen, NP0002). Novex™ Sharp Pre-stained protein standard (Invitrogen, LC5800) was used as marker to estimate molecular weight of the proteins. The gel was stained using Pierce Silver Stain kit (Thermo, 24612) as per manufacturer’s protocol and imaged using ProteinSimple FluorChem E imaging system.
4
Western Blot Protocol for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were mixed with 4 x LDS sample buffer (ThermoFisher NP0007) and 0.1M DTT. Samples were loaded onto a 4–12% Bis-Tris gel (Invitrogen NP0321BOX) in 1x MOPs buffer and run at 160V for 1 hour. The gel was transferred to a nitrocellulose membrane using the wet transfer method in 1x transfer buffer (25mM Tris base, 192mM glycine, 20% methanol) at 400mA for 75 minutes at 4°C. The membrane was blocked in a blocking buffer (5% non-fat milk powder in 1x Tris buffer saline with 0.1% Tween 20) for at least 60 minutes. Primary antibodies were diluted in blocking buffer and incubated with membranes overnight at 4°C. Membranes were washed 3 × 20 minutes with 1 x TBST, incubated for 1 hour at 25°C with secondary HRP-conjugated antibodies against rabbit IgG (Cell Signaling Technology, 7074S), washed again 3× 20 minutes with 1x TBST, and developed using ECL Western Blotting Detection Reagents (Cytiva) and Amersham Hyperfilm ECL (Cytiva) using an automated M35 X-OMAT Processor developer (Kodak).
5
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated at 85% confluency and allowed to grow overnight. The following day, the cells were collected and protein was extracted on ice using cell lysis buffer (cell signaling, #9803) with protease and phosphatase supplements and protein amount was quantified using BCA as previously described. A total of 300 µg of protein was mixed with prewashed magnetic beads (Thermo Scentific, #88802) and primary antibody (concentration used as per the manufacturer’s recommendation) and incubated overnight at 4 °C. The following day, beads were washed again to remove unbound antibody, boiled with 4X SDS (Thermo Scientific, #NP0007) and analyzed using western blot.
6
Protein Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Optical density was measured and a total of 6 OD600 units was collected for each time point. Cells were pelleted and stored at −80°C. Cell lysates were prepared by adding 150 μl lysis buffer (1.85 M NaOH, 1/100 B-ME, 1/50 protease inhibitors) followed by a 10-minute incubation on ice. After incubation, 150 μl of 50% TCA (Sigma-Aldrich T9159) was added. After a 10-minute incubation at 4°C, samples were spun for 15 seconds at 15000 RPM and supernatant was aspirated. The remaining pellet was washed with one ml acetone, briefly spun, and acetone aspirated. 100 μl of 2x sample buffer (ThermoFisher NP0007) with 10% B-ME were added to the protein pellets, mixed well, and boiled for 5 minutes.
7
Protein Extraction and In-Gel Digestion
8
Antibody-Antigen Complex Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An antibody-antigen complex was prepared by adding 100 µg of protein lysate and primary antibody in RIPA buffer to a total volume of 1 ml, then incubated overnight at 4 °C on a rotary tube mixer. The following day, 50 µl of protein-G magnetic beads (Millipore, LSKMAGG10) were dispensed into a 1.5 ml collection tube and placed on a magnetic rack (Millipore, 20–400). The storage substrate was removed and the beads were washed twice in 500 µl PBS with 0.1% Tween-20 (PBST). The mixture was briefly vortexed and centrifuged between washes and the supernatant was discarded. Next, the antibody-antigen complex was mixed with the magnetic beads and incubated overnight on a rotary tube mixer at 4 °C. The next day, the mixture was briefly centrifuged and washed 3 times in PBST. The elution was resuspended in 25 µl RIPA buffer with reducing agent (ThermoFisher, NP0004) and sample buffer (ThermoFisher, NP0007). Next, the samples were denatured by heating at 95 °C for 10 and loaded into 4–12% Bis-Tris pre-cast protein gels for SDS-PAGE.
9
Nebulized SARS-CoV-2 Nanobody Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nanobody was nebulized with a portable mesh nebulizer (Philips, InnoSpire Go) producing 2-5 μm particles at a final concentration of 0.4 mg ml−1. The resulting aerosol was collected by condensation into a 50-ml tube cooled on ice. Pre- and post-nebulization samples were analysed by NuPAGE gel and visualized by InstantBlue staining. SARS-CoV-2 surrogate virus neutralization test was also performed to compare the neutralization potency of pre- and post-nebulization samples. For thermostability tests, nanobodies supplemented with loading buffer (Thermo Fisher Scientifc, NP0007) and β-mercaptoethanol were heated at 98 °C for 10 min and then analysed on a NuPAGE gel and visualized by InstantBlue staining.
10
SDS-PAGE Protein Denaturation and Separation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A volume containing 5 µg of protein, 1 x sample buffer (ThermoFisher, NP0007), 1 x reducing agent (ThermoFisher, NP0004) and ddH2O were denatured by heating at 95 °C for 5 minutes (or 100 °C for 15 minutes for TTR monomer detection). The sample mixture was cooled on ice for 5 minutes before being briefly centrifuged. Samples and protein ladder (ThermoFisher, 26634) were loaded into 4–12% Bis-Tris pre-cast protein gels (ThermoFisher, NP0335) or 12% Bis-Tris pre-cast protein gels for TTR controls (ThermoFisher, NP0341), in MOPS running buffer supplemented with 1:400 antioxidant (ThermoFisher, NP0005). Electrophoresis (Biorad, Mini-protean) was set to 150 V for 1.5 hours at room temperature.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!