Transfected HEK293 cell lysate was incubated with 500 μg of peptide F (Proteintech) or control peptide (Proteintech) overnight at 4°C. The sample was cross-linked with 2 mM DTSSP (Thermo Scientific) for 1 hour at room temperature. The sample was precleared by adding 10 μg of mouse immunoglobulin G and 20 μl of protein A/G agarose and incubated at 4°C for 2 hours. After centrifugation at 1000g for 5 min, the supernatant was incubated with 5 μg of anti-DDK antibodies (OriGene) overnight at 4°C. Protein A/G PLUS-Agarose beads (40 μl) (Santa Cruz Biotechnology) were added and incubated for 2 hours at 4°C, then centrifuged for 5 min at 1000g at 4°C. The pellet was washed three times with IP lysis buffer (Thermo Scientific), followed by addition of 80 μl of 2× sample buffer (Santa Cruz Biotechnology). The sample was boiled for 5 min and then centrifuged for 5 min at 1000g at 4°C. The supernatant was loaded onto a 15% SDS-PAGE gel and probed with antibodies to peptide F (nonreducing condition), DDK, and CD59 (both in reducing condition with 5% β-mercaptoethanol).
Sample buffer
Manufactured by Santa Cruz Biotechnology
Sample buffer is a liquid solution used to prepare samples for analysis or testing in a laboratory setting. It is designed to maintain the stability and integrity of the sample during various analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Peptide f, by Proteintech (2 mentions)
Anti ddk antibody, by OriGene (2 mentions)
Dtssp, by Thermo Fisher Scientific (2 mentions)
Control peptide, by Proteintech (2 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (2 mentions)
Tla100.2 rotor, by Beckman Coulter (2 mentions)
Recombinant smn, by Enzo Life Sciences (2 mentions)
6 protocols using sample buffer
1
Peptide F-Protein Interaction Assay
2
Peptide F-Protein Interaction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected HEK293 cell lysate was incubated with 500 μg of peptide F (Proteintech) or control peptide (Proteintech) overnight at 4°C. The sample was cross-linked with 2 mM DTSSP (Thermo Scientific) for 1 hour at room temperature. The sample was precleared by adding 10 μg of mouse immunoglobulin G and 20 μl of protein A/G agarose and incubated at 4°C for 2 hours. After centrifugation at 1000g for 5 min, the supernatant was incubated with 5 μg of anti-DDK antibodies (OriGene) overnight at 4°C. Protein A/G PLUS-Agarose beads (40 μl) (Santa Cruz Biotechnology) were added and incubated for 2 hours at 4°C, then centrifuged for 5 min at 1000g at 4°C. The pellet was washed three times with IP lysis buffer (Thermo Scientific), followed by addition of 80 μl of 2× sample buffer (Santa Cruz Biotechnology). The sample was boiled for 5 min and then centrifuged for 5 min at 1000g at 4°C. The supernatant was loaded onto a 15% SDS-PAGE gel and probed with antibodies to peptide F (nonreducing condition), DDK, and CD59 (both in reducing condition with 5% β-mercaptoethanol).
3
Purification and Characterization of SMN-Bound Ribosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purification of 80S ribosomes was performed from NSC-34 depleted from SMN using CRISPR-Cas9 technology (11 (link)). The lysate was treated with RNase I (7.5 Units /1 Abs260 lysate) at RT for 45 min and analyzed by sucrose gradient (10-40%) (65 (link)). The fraction corresponding to the 80S was collected and 2mM DTT was added. After centrifugation at 90,000 rpm for 4 h using a TLA100.2 rotor (Beckmann) the pellet was resuspended in 10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 150 mM NaCl, 2 mM DTT, 100 μg/mL cycloheximide and stored at −80°C. Ribosome concentration was calculated as in (66 (link)). Recombinant SMN was purchased (ENZO) and incubated with ribosomes for 2 h at 4°C. SMN bound ribosomes were purified from unbound SMN by ultracentrifugation at 90,000 rpm for 4 h using the TLA100.2 rotor on 30% sucrose cushion. The supernatant was kept for protein purification by using the chloroform/methanol protocol and the pellet was directly dissolved in sample buffer (Santa Cruz), heated at 99°C for 10 min and resolved by SDS-PAGE. SMN and RPL26, were detected using primary and secondary antibodies described above.
4
Purification and Characterization of SMN-Bound Ribosomes
5
Puromycin Treatment Impacts Ribosome Composition
6
Puromycin Treatment Impacts Ribosome Composition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Control MCF7 cells and MCF7 cells treated with puromycin 100 μM for 1h were lysed according to (36 (link)). Active ribosomes were isolated using RiboLace kit (IMMAGINA Biotechnology). Proteins were extracted from beads using sample buffer (Santa Cruz). Five μL of lysate were kept as input. Proteins were resolved using SDS-PAGE and western blotting as in (36 (link)).
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!