The largest database of trusted experimental protocols

Nap 10 sephadex g 25 column

Manufactured by GE Healthcare
Sourced in Germany

The NAP-10 Sephadex G-25 column is a disposable, pre-packed column used for desalting and buffer exchange of small molecules, proteins, and peptides. The column is filled with Sephadex G-25 resin, which provides efficient separation and removal of salts, buffers, and other low molecular weight compounds from sample solutions.

Automatically generated - may contain errors

6 protocols using nap 10 sephadex g 25 column

1

Calcein Leakage Assay for Lipid Vesicles

Check if the same lab product or an alternative is used in the 5 most similar protocols
Purified calcein-loaded LUVs were mixed with D-dfTAT in presence or absence of UNC7938 (1–100 μM) at a 1:50 peptide: lipid ratio for 1 h at room temperature in 100 mM NaCl, 10 mM NaH2PO4 pH5.5. UNC7938 stock solution Samples were centrifuged for 1 min at 4,000 rpm. To measure the amount of leaked calcein and separate soluble liposomes from released calcein, the supernatants were purified using an illustra NAP-10 Sephadex G-25 column (GE Healthcare) (the elution volumes of liposomes and free calcein were determined independently with pure samples). Fractions were collected in a 96-well plate and the fluorescence of calcein was measured using a Promega GloMax-Multi plate reader (Ex 490nm, Em 520–560nm). Note, since UNC7938 stock is dissolved in 100% DMSO. The control of D-dfTAT+ 0.1% DMSO (concentration of DMSO in the UNC7938 incubations) with liposomes was performed as a control. 100% leakage was established by treating liposomes with the detergent Triton X-100.
+ Open protocol
+ Expand
2

Conjugation of scFvs to Tetrazine-Peptide

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 25

This example aimed to demonstrate that three scFvs could be conjugated to the three PEG12-maleimide linking arms based on tetrazine-peptide 2. Prior to conjugation with the tetrazine-peptide 2 that had three PEG12-maleimide linking arms, 1F10 scFv was incubated with DTT at a molar ratio of 2:1 ([DTT]:[scFv]) at 25° C. for 4 hours with gentle shaking to keep its C-terminal cysteine in reduced form. Subsequently, the buffer of reduced 1F10 scFv was exchanged to maleimide-SH coupling reaction buffer (100 mM sodium phosphate, pH 7.0, 50 mM NaCl and 5 mM EDTA) by using an NAP-10 Sephadex G-25 column (GE Healthcare). After the reduction and buffer exchange, the conjugation to the tetrazine-peptide 2 having three PEG12-maleimide linking arms was conducted overnight at 4° C. at a molar ratio of 1:4 ([linker]:[Protein]).

+ Open protocol
+ Expand
3

Conjugation of scFv to Tetrazine-Peptide

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 26

This example aimed to demonstrate that three scFvs could be conjugated to the three PEG12-maleimide linking arms based on tetrazine-peptide 2. Prior to conjugation with the tetrazine-peptide 2 that had three PEG12-maleimide linking arms, 1F10 scFv was incubated with DTT at a molar ratio of 2:1 ([DTT]:[scFv]) at 25° C. for 4 hours with gentle shaking to keep its C-terminal cysteine in reduced form. Subsequently, the buffer of reduced 1F10 scFv was exchanged to maleimide-SH coupling reaction buffer (100 mM sodium phosphate, pH 7.0, 50 mM NaCl and 5 mM EDTA) by using an NAP-10 Sephadex G-25 column (GE Healthcare). After the reduction and buffer exchange, the conjugation to the tetrazine-peptide 2 having three PEG12-maleimide linking arms was conducted overnight at 4° C. at a molar ratio of 1:4 ([linker]:[Protein]).

+ Open protocol
+ Expand
4

Calcein Leakage Assay for Peptide-Lipid Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols
Purified calcein-loaded LUVs were mixed with different peptides at a 1:50 peptide: lipid ratio for 1 h at room temperature in 100 mM NaCl, 10 mM NaH2PO4 pH5.5. Samples were centrifuged for 1 min at 4,000 rpm. To measure the amount of leaked calcein and to separate soluble liposomes from released calcein, the supernatants were purified using an illustra NAP-10 Sephadex G-25 column (GE Healthcare) (the elution volumes of liposomes and free calcein were determined independently with pure samples). Fractions were collected in a 96-well plate and the fluorescence of calcein was measured using a Promega GloMax-Multi plate reader (Ex 490nm, Em 520–560nm). The percent leakage was calculated using the following equation:
%Leakage=100×Flt-Fl0Flmax-Fl0 where Flt is the free calcein fluorescence intensity of a sample at a specific peptide:lipid ratio measured after 1 h, Fl0 is the free calcein fluorescence intensity of an untreated sample and Flmax is the free calcein fluorescence intensity of a sample after treatment with 0.2 % Triton X-100.
+ Open protocol
+ Expand
5

Calcein Leakage Assay for Liposome Permeabilization

Check if the same lab product or an alternative is used in the 5 most similar protocols
A calcein-leakage assay was employed to evaluate the permeabilization of liposomes by dfTAT. For this assay, LUVs of different lipid compositions containing 60 mM calcein in their lumen were prepared. LUVs were mixed with dfTAT and fTAT at different peptide:lipid ratios in phosphate buffer (100 mM NaCl, 10 mM NaH2PO4 pH 5.5). After 60 min, the amount of free calcein was established by purifying the supernatants obtained using an illustra NAP-10 Sephadex G-25 column (GE Healthcare). The supernatants obtained after low speed centrifugation were purified to exclude soluble liposomes. The fluorescence from free calcein, from the fractions collected, was measured using the blue channel (Ex 490 nm, Em 520–560 nm) of a Promega GloMax-Multi plate reader (Promega). The extent of liposomal lumen content release or % leakage was determined according to the following equation:
%Leakage=100×FltFl0FlmaxFl0 Where Flt is the free calcein fluorescence intensity of a sample at a specific peptide:lipid ratio measured after 60 min, Fl0 is the free calcein fluorescence intensity of a untreated sample and Flmax is the free calcein fluorescence intensity of a sample after treatment with 0.2 % Triton X-100.
+ Open protocol
+ Expand
6

Functionalization of MWCNT with CS-ODN

Check if the same lab product or an alternative is used in the 5 most similar protocols
25 µl of a 1 mM NH2-CS-ODN solution in water was mixed with 7.5 µl NaHCO3-buffer (pH 8.6, 1 M) and 25 µl DSS (10 mg/ml in DMSO). Excess DSS was separated using an illustra™ NAP™-10 sephadex™ G-25 column (GE Healthcare, Munich, Germany) and activated ODN were concentrated by Amicon® Ultra centrifugal filters (3000 MWCO; Merck, Darmstadt, Germany). 0.5 nmol of the activated NH2-CS-ODN were mixed with 50 µg MWCNT-NH2 in 0.5 ml PBS and shaken at room temperature overnight followed by the removal of the excess CS-ODN.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!