For each replicate experiment, cardiac mitochondria isolated from 3–5 pooled hearts of each genotype were lysed on ice for 30 min in 1X RIPA buffer (EMD Millipore #20-188) supplemented with 1X protease inhibitors (Sigma-Aldrich #S8830-20TAB), and lysates were cleared by centrifuging at 14000g for 10min at 4°C. Protein concentration was determined by bicinchoninic acid assay (BioWORLD #20831001). 2500μg of cleared mitochondrial lysate were fractionated by gel filtration using fast protein size-exclusion liquid chromatography (AKTA Pure FPLC; GE Healthcare), using a Superdex 200 Increase 10/300 column (Sigma-Aldrich, #GE28-9909-44) equilibrated in 1X PBS, at a flow rate of 0.5mL/min. 0.5mL protein fractions were collected, concentrated to 75μL with 3kD molecular weight cutoff AMICON Ultra-0.5 centrifugal filter devices (EMD Millipore #UFC500396) following the manufacturer’s instructions. Concentrated protein fractions were used for western blotting under reducing conditions as described below. Molecular weights of FPLC fractions were calibrated using gel filtration markers (Sigma-Aldrich #MWGF1000).
Superdex 200 increase 10 300 column
Manufactured by Merck Group
Sourced in United States
The Superdex 200 Increase 10/300 column is a size-exclusion chromatography column designed for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. The column features a stable, rigid matrix that provides high resolution and reproducible results. Its core function is to allow for the separation and purification of target molecules based on their size and molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Ufc500396, by Merck Group (1 mentions)
Akta pure fplc, by GE Healthcare (1 mentions)
Mwgf1000, by Merck Group (1 mentions)
Carbonic anhydrase, by Merck Group (1 mentions)
Gel filtration calibration kit, by Cytiva (1 mentions)
Cytochrome c, by Merck Group (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
2 protocols using superdex 200 increase 10 300 column
1
Cardiac Mitochondria Fractionation by FPLC
2
Characterizing TNFα-Ozoralizumab Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ozoralizumab or adalimumab (0.65 nmol) was mixed with 0.072, 0.217, 0.65, or 1.95 nmol of TNFα trimer in 250 μL of PBS. Each mixture was incubated overnight at 4°C and then loaded onto a Superdex 200 Increase 10/300 column equilibrated with PBS. Representative results from two independent experiments are shown in Figure 1
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!