Total extracts from 2x107 cells were immunoprecipitated with anti-Drp1 antibody (BD Transduction Laboratories, Erembodegem, Belgium) according to standard protocols. Briefly, extracts were pre-cleared with protein A/G agarose beads, incubated o.n. at 4°C with 5µg of antibody and subsequently with protein A/G agarose beads for 2 hours. Beads were washed 3 times with 1X IP high buffer (Active Motif, Carlsbad, US) supplemented with 1 mg/ml BSA and 3 times with 1X wash IP high buffer. Immunoprecipitated proteins were eluted for 10 minutes at 70°C with NuPAGE-LDS Sample Buffer 2X (Thermo Fischer Scientific, Carlsbad, US), supplemented with 50 mM DTT and further investigated by immunoblotting.
Ip high buffer
Manufactured by Active Motif
Sourced in United States
1X IP high buffer is a laboratory reagent used in the preparation of samples for immunoprecipitation (IP) experiments. It is designed to maintain the optimal pH and ionic strength for the capture and recovery of target proteins from cell or tissue lysates.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ip high buffer
1
Drp1 Protein Immunoprecipitation and Analysis
2
Co-Immunoprecipitation of FECH, RNAP1, and CSB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates or subcellular fractions from 0.3–1 × 107 cells, were immunoprecipitated with anti-FECH (S. Cruz, sc-377377ac), anti-RNAP1 (S. Cruz, sc-48385ac) or anti-CSB (A301-345A Bethyl) antibodies conjugated with A/G agarose beads according to standard protocols (54 (link)). Briefly, extracts were incubated overnight at 4°C with 15 μg of antibody-conjugated beads. Beads were washed three times with 1X IP high buffer (Active Motif) supplemented with 1 mg/ml BSA and three times with 1× IP high buffer. Immunoprecipitated proteins were eluted for 10 min at 70°C with NuPAGE-LDS Sample Buffer 2× (Thermo Fischer Scientific), supplemented with 50 mM DTT and further investigated by immunoblotting.
For the two-step co-immunoprecipitation (TIP), chromatin enriched fractions were first immunoprecipitated with anti-FECH antibody (S. Cruz, sc-377377ac) following the conditions described above. The immunoprecipitated proteins were eluted three times at RT with Ferrochelatase (A-3) blocking peptide (S.Cruz, sc-377377 P) and subsequently immunoprecipitated with anti-RPA194 antibody (S. Cruz, sc-48385ac). Final elution was performed for 10 min at 70°C with NuPAGE-LDS Sample Buffer 2× (Thermo Fischer Scientific) supplemented with 50 mM DTT.
For the two-step co-immunoprecipitation (TIP), chromatin enriched fractions were first immunoprecipitated with anti-FECH antibody (S. Cruz, sc-377377ac) following the conditions described above. The immunoprecipitated proteins were eluted three times at RT with Ferrochelatase (A-3) blocking peptide (S.Cruz, sc-377377 P) and subsequently immunoprecipitated with anti-RPA194 antibody (S. Cruz, sc-48385ac). Final elution was performed for 10 min at 70°C with NuPAGE-LDS Sample Buffer 2× (Thermo Fischer Scientific) supplemented with 50 mM DTT.
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!