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 .
Np0336box
Manufactured by Merck Group
The NP0336BOX is a laboratory equipment product manufactured by Merck Group. It serves as a storage and transport container for various laboratory samples and materials. The core function of this product is to provide a secure and organized way to handle and protect sensitive laboratory items.
Automatically generated - may contain errors
2 protocols using np0336box
1
Western Blot for Hematopoietic Stem Cells
2
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 .
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!