MST样品标记说明书RED-NHS

MST样品标记说明书RED-NHS

Cat Nr: L001

Monolith NT Protein Labeling Kit RED-NHS

Microscale Thermophoresis Grade

Kit for 1h labeling of proteins with the RED fluorescent dye NT-647-NHS

for use in Microscale Thermophoresis

CONTENT AND

STORAGE Monolith NT Protein Labeling Kits are shipped on at room temperature . Store at 4°C upon arrival. Each kit contains material sufficient for 4 protein

labeling reactions. Depending on the amount of protein used, enough material

for approximately 1000 Microscale Thermophoresis can be prepared.

4* 25µg NT-647-NHS dye

4* Spin Column A, Buffer Exchange

4* Gravity Flow Column B, Purification

2* Adapter 15 ml Falcons

4* Labeling Buffer

Expiry Date: See Kit cover

ADDITIONAL MATERIAL

REQUIRED

Variable speed benchtop microcentrifuge 1.5 -2 ml microcentrifuge collection tubes Exchange buffer (buffer of choice e.g. PBS) 100% DMSO Aq.dest.

User Manual Monolith NT Protein Labeling Kit V011 Date Feb 2012 1

MST样品标记说明书RED-NHS

User Manual Monolith NT Protein Labeling Kit V011 Date Feb 2012 2

MST样品标记说明书RED-NHS

PROTEIN LABELING PROCEDURE

The Monolith NT Protein Labeling Kit provides convenient means for labeling small amounts (2 – 20 µM) of purified protein with our NT-647-NHS fluorescent dye. This kit has been optimized for labeling proteins with molecular weights higher than 10 kDa, and contains everything needed to perform four labeling reactions and to separate the resulting conjugates from excess dye. Convenient gravity flow and spin

columns are used to purify the labeled protein with higher recovery yields (60-90 %) depending primarily on the molecular weight of the starting material. Labeling and purification can be completed in less than 60 minutes.

The NT-647-NHS reactive dye contains NHS-ester chemistry, which reacts efficiently with primary amines of proteins to form highly stable dye-protein-conjugates. NT-647-NHS dye labeled proteins show fluorescence excitation and emission maxima of approximately 650 and 670 nm, respectively.

STEP A

BUFFER EXCHANGE

The labeling step requires that the protein is dissolved in a suitable labeling buffer at the correct pH. The purified protein should be in a buffer that does not contain primary amines (e.g., ammonium ions, Tris, glycine,

ethanolamine, triethylamine, glutathione) or imidazole. All of these

substances will significantly reduce protein labeling. Also, partially purified

protein samples, or protein samples containing carriers like BSA, will not be

labeled properly and should not be used.

1. Add 3.0 ml aq. dest. to the vials containing the labeling buffer salt

2. Invert Spin Column-A to suspend slurry

3. Twist off bottom (twist slightly in both directions)

4. Loose the cap of the column

5. Place column in 1.5 -2 ml microcentrifuge collection tube

6. Centrifuge at 1.500xg for 1 minute to remove excess liquid

7. Add 300 µl of labeling buffer and repeat step 5 (repeat 3 times)

8. Place 40-100 µl protein in the center of the resin. Be careful not to

disturb the resin or allow the sample to flow around the resin bed.

9. Place the sample in new microcentrifuge collection tube and centrifuge

at 1500xg for 2 minutes

User Manual Monolith NT Protein Labeling Kit V011 Date Feb 2012 3

MST样品标记说明书RED-NHS

STEP B

LABELING

For protein labeling 1. Adjust the protein concentration to 2-20 µM using the Labeling Buffer (maximum of 250µl protein solution can be used per labeling procedure). For Calculation of the molarity of your sample use the

following equation:

c(Protein) [Mol/l] = c(Protein) [mg/ml] / MW (Protein) [Da]

2. Add 50 µl of 100% DMSO to the solid fluorescent dye (i.e. approx. 650µM solution)*

3. Mix the dye thoroughly by vortexing and make sure that all dye is solved

4. Adjust the concentration of the dye to 2-3 fold concentration of the protein by using the labeling buffer

5. Mix Protein and Dye in a 1:1 ratio (500 µl maximum volume per gravity flow column-B)

6. Incubate for 30 minutes at room temperature in the dark

7. In the meantime, prepare Step C

*We do not recommend using the dye for more than a few hours after suspending it. Freeze the dye if you intent to use it again.

STEP C

PURIFICATION For optimal results use in Microscale Thermophoresis, unreacted “free” dye needs to be eliminated. The kit is optimized for this. The purity can be

monitored by measuring the ratio of protein to dye (e.g. spectrocopically by measuring absorption at 280nm for protein and 650 nm for the dye, Molar absorbance: 250.000 M-1cm-1) after clean-up procedure.

1. Remove the top cap of Column-B and pour off the column storage solution

2. Remove the bottom cap and place in a 15 ml tube (Adapter supplied)

3. Equilibrate and wash Column B by adding 8 ml of your final protein storage / analysis buffer of choice (Flow through by gravity flow).

4. Add maximum 500 µl of labeling reaction to the center of Column B. Let sample enter the bed completely (when using less than 500 µl, using your final storage/analysis buffer)

5. Discard the flow through

6. Place in a new 15 ml collection tube

7. Add 600µl of storage/analysis buffer and collect the eluate. The first few µl will not contain protein and can be discarded.

Tip: By collecting aliquots you can increase the sample concentration. If you use a protein with MW of 10- …… 此处隐藏：5964字，全部文档内容请下载后查看。喜欢就下载吧 ……