WB was performed as described previously38 (link). Approximately 20,000 SLAM HSCs were sorted into 250 μl PBS containing 20% trichoracetic acid (TCA). The concentration of TCA was adjusted to 10% after sorting. Extracts were incubated on ice for 30 min and centrifuged at 13,000 rpm for 10 min at 4°C. Precipitates were washed in pure acetone (Fisher scientific, A18-4) twice and the dried pellets were solubilized in 9 M urea, 2% Triton X-100, and 1% DTT together with 1X LDS buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. NativePAGE™ 4-16% Bis-Tris protein gel was used to detect MPL aggregates in HSCs as manufacturer’s instructions39 (link) (Invitrogen, BN1002BOX). Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096). Antibodies and reagents used are in Supplementary Table 1 and 2 .
A18 4
Manufactured by Thermo Fisher Scientific
The A18-4 is a laboratory centrifuge designed for general-purpose applications. It features a fixed-angle rotor with a capacity of 4 tubes. The centrifuge provides reliable and consistent performance for a variety of sample preparation and separation tasks.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto chemiluminescence kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bn1002box, by Thermo Fisher Scientific (2 mentions)
Np0336box, by Merck Group (2 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
A451 4, by Thermo Fisher Scientific (1 mentions)
Cary 60 uv vis instrument, by Agilent Technologies (1 mentions)
Pierce c18 spin column, by Thermo Fisher Scientific (1 mentions)
V5280, by Promega (1 mentions)
I1149, by Merck Group (1 mentions)
6 protocols using a18 4
1
Western Blot for Hematopoietic Stem Cells
2
Multistep Droplet Drying for Optical and UV-Vis Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples for optical microscopy and UV–vis
transmission measurements were prepared using a multistep droplet
drying/mixing process. First, a stock solution of the largest BBCP
studied (N400) was prepared at 100 mg/mL in toluene. Glass coverslips
were sequentially rinsed in toluene, acetone (Fisher Chemical A18-4),
and isopropanol (Fisher Chemical A451-4) before being dried using
a nitrogen gun. To achieve the target concentration between slides,
a series of droplets were added to the center of the slide and allowed
to dry before the addition of a final droplet with the required amount
of solution for the target concentration. To aid in mixing, a square
“well” was made by placing two additional coverslips
on the side of the final droplet before covering with the top coverslip
to enforce a 1 mm gap. The encased droplet was gently tapped until
the sample was fully dissolved, before removal of the coverslip spacers
to obtain the final gap size (determined by capillary forces). Optical
microscope images were obtained under diffuse (ring) light against
a dark background (anodized aluminum). Transmission spectra were collected
using a Cary 60 UV–vis instrument (Agilent).
transmission measurements were prepared using a multistep droplet
drying/mixing process. First, a stock solution of the largest BBCP
studied (N400) was prepared at 100 mg/mL in toluene. Glass coverslips
were sequentially rinsed in toluene, acetone (Fisher Chemical A18-4),
and isopropanol (Fisher Chemical A451-4) before being dried using
a nitrogen gun. To achieve the target concentration between slides,
a series of droplets were added to the center of the slide and allowed
to dry before the addition of a final droplet with the required amount
of solution for the target concentration. To aid in mixing, a square
“well” was made by placing two additional coverslips
on the side of the final droplet before covering with the top coverslip
to enforce a 1 mm gap. The encased droplet was gently tapped until
the sample was fully dissolved, before removal of the coverslip spacers
to obtain the final gap size (determined by capillary forces). Optical
microscope images were obtained under diffuse (ring) light against
a dark background (anodized aluminum). Transmission spectra were collected
using a Cary 60 UV–vis instrument (Agilent).
3
TCA Precipitation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as described previously (Xu et al., 2020 (link)). Approximately 5 × 105 DN3 cells were sorted directly into 250 μl PBS containing 20% trichoracetic acid (TCA). The concentration of TCA was adjusted to 10% after sorting and cells were incubated on ice for 30 min before centrifugation at 13,000 rpm for 10 min at 4°C. Precipitates were washed twice with pure acetone (Fisher scientific, A18-4) and solubilized in 9 M urea, 2% Triton X-100, and 1% dithiothreitol (DTT) in 1 x LDS sample buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096). Antibodies and reagents used are in Appendix 1. The original western blot images are in source data files.
4
Western Blot for Hematopoietic Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WB was performed as described previously38 (link). Approximately 20,000 SLAM HSCs were sorted into 250 μl PBS containing 20% trichoracetic acid (TCA). The concentration of TCA was adjusted to 10% after sorting. Extracts were incubated on ice for 30 min and centrifuged at 13,000 rpm for 10 min at 4°C. Precipitates were washed in pure acetone (Fisher scientific, A18-4) twice and the dried pellets were solubilized in 9 M urea, 2% Triton X-100, and 1% DTT together with 1X LDS buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. NativePAGE™ 4-16% Bis-Tris protein gel was used to detect MPL aggregates in HSCs as manufacturer’s instructions39 (link) (Invitrogen, BN1002BOX). Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096). Antibodies and reagents used are in Supplementary Table 1 and 2 .
5
Porcine Ovary Proteomics Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two technical replicates of four porcine ovaries from four separate animals (two ovaries per direction) were used to generate seven slices each in the proteomics analysis. 50 µg of protein was precipitated with eight volumes of cold acetone (Fisher, A18–4) and one volume of trichloroacetic acid (Sigma, T9159-250G) overnight at −20 °C. After washing the pellet with ice-cold acetone, resulting protein pellet was resuspended in 50 µL 8 M urea (Invitrogen, 15505-035) in 400 mM ammonium bicarbonate (Fisher, A643-500), pH 7.8, reduced with 4 mM dithiothreitol (Sigma, 10197777001) at 50 °C for 30 min., and cysteines were alkylated with 18 mM iodoacetamide (Sigma, I1149) in the dark for 30 min. The solution was then diluted to <2 M urea and trypsin (Promega, V5280) was added at final trypsin:protein ratio of 1:50 prior to overnight incubation at 37 °C with shaking. The resulting peptides were desalted using solid phase extraction on a Pierce C18 Spin column (Thermo, 89873) and eluted in 80 mL of 80% acetonitrile (ACN) (Thermo, 51101) in 0.1% formic acid (FA) (Fisher, LS118). After lyophilization, peptides were reconstituted with 5% ACN in 0.1% FA.
6
Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as described previously (Xu et al., 2020) (link). Approximately 5 × 10 5 DN3 cells were sorted directly into 250 μl PBS containing 20% trichoracetic acid (TCA).
The concentration of TCA was adjusted to 10% after sorting and cells were incubated on ice for 30 min before centrifugation at 13,000 rpm for 10 min at 4 °C. Precipitates were washed twice with pure acetone (Fisher scientific, A18-4) and solubilized in 9 M urea, 2% Triton X-100, and 1% dithiothreitol (DTT) in 1 x LDS sample buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4-12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies. Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096) . Antibodies and reagents used are in Supplementary Table 1 and3
The concentration of TCA was adjusted to 10% after sorting and cells were incubated on ice for 30 min before centrifugation at 13,000 rpm for 10 min at 4 °C. Precipitates were washed twice with pure acetone (Fisher scientific, A18-4) and solubilized in 9 M urea, 2% Triton X-100, and 1% dithiothreitol (DTT) in 1 x LDS sample buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4-12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies. Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096) . Antibodies and reagents used are in Supplementary Table 1 and3
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!