After pooling and sample clean-up by solid phase extraction (SPE strata-X-C, Phenomenex), the sample pool was subjected to peptide IEF-IPG (isoelectric focusing by immobilized pH gradient) in pI range 3–10 (350 µg). Freeze dried peptide samples were dissolved in 250 µL rehydration solution containing 8 M urea and 1% IPG pharmalyte pH 3–10 and allowed to adsorb to the 24 cm linear gradient IPG strip by swelling overnight. Peptides were focused on the IPG strip as described in Branca et al., 2014 (link). After focusing, the peptides were passively eluted into 72 contiguous fractions with MilliQ water using an in-house constructed IPG extractor robotics (GE Healthcare Bio- Sciences AB, prototype instrument) into a 96-well plate (V-bottom, Corning product #3894), which were then dried in a SpeedVac. The resulting fractions were freeze dried and kept at −20°C.
V bottom
Manufactured by Corning
Sourced in Sweden, United States
The V-bottom is a versatile piece of lab equipment used for a variety of applications. Its distinctive V-shaped design provides a stable base and facilitates the mixing and handling of liquids and samples during laboratory procedures.
Automatically generated - may contain errors
4 protocols using v bottom
1
Peptide Fractionation by IEF-IPG
2
Cytoflex Flow Cytometry Standardization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Detector gain settings were set to “Recommended” and threshold to “Automatic” for all tests, to use the instrument's QC settings based on the daily SST. Laminar flow conditions were maintained with the Cytoflex Sheath Fluid (Beckman Coulter, cat. no. B51503) and all samples were acquired from 96-well plates with V-bottom (Corning, Amsterdam, NL, cat. no. 3897). Acquisition of one well was considered equivalent to one test (n = 1).
3
Peptide Fractionation by IEF-IPG
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After pooling and sample clean-up by solid phase extraction (SPE strata-X-C, Phenomenex), the sample pool were subjected to peptide IEF-IPG (isoelectric focusing by immobilized pH gradient) in pI range 3–10 (350 μg). Freeze dried peptide samples were dissolved in 250 μl rehydration solution containing 8 m urea and 1% IPG pharmalyte pH 3–10 and allowed to adsorb to the 24 cm linear gradient IPG strip by swelling overnight. Peptides were focused on the IPG strip as described in (21 (link)). After focusing, the peptides were passively eluted into 72 contiguous fractions with MilliQ water using an in-house constructed IPG extractor robotics (GE Healthcare Bio- Sciences AB, Uppsala, Sweden, prototype instrument) into a 96-well plate (V-bottom, Corning, Hickory, North Carolina product #3894), which were then dried in a SpeedVac. The resulting fractions were freeze dried and kept at −20 °C.
4
Peptide Fractionation via IEF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each labeled peptide pool (200 μg) was dissolved in 250 μl of rehydration solution (8 M urea, 1% pharmalyte for pH range 3–10 from GE Healthcare, Little Chalfont, UK), which was then used to re-swell an immobilized pH gradient gel-strip (GE Healthcare) pH 3–10. For the IEF in the 3.7–4.9 pH range, 200 μg of labeled peptide pool were dissolved in 150 μl of 8 M urea, and this solution was used to rehydrate a sample gel bridge (pH 3.7) overnight. The 3.7–4.9 immobilized pH gradient strip was rehydrated in 8 M urea, 1% pharmalyte for pH range 2.5–5 (GE Healthcare). All IEFs were run on an Ettan IPGphor (GE Healthcare) until at least 150 kVh for the 3–10 range and until at least 250 kVh for the 3.7–4.9 range (~1 day running time in either case). After focusing was complete, a well-former with 72 wells was applied onto each strip, and liquid-handling robotics (GE Healthcare prototype) added MilliQ water and, after 3 × 30 min incubation/transfer cycles, transferred the 72 fractions into a microtiter plate (96 wells, V-bottom, Corning, Lowell, MA, USA, Cat. No. 3894), which was then dried in a SpeedVac.
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!