Labelling Kits Centrifugation-Based Rapid Desalting Column (5KD)

Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.)
This desalination column is specifically designed for protein sample desalination and buffer replacement. It uses a gel filtration medium with a molecular weight cut-off of 5KD. This product is a pre-filled column and is used in conjunction with PBS buffer, suitable for removing small molecule impurities (such as free dyes, salt ions, etc.) or for rapid buffer replacement.
1. Preparation Work
1.1 Remove the desalination column and carefully remove the bottom sealing plug and the top cover.
1.2 Place the column into the corresponding collection tube.
2. Balance the columns
2.1 Filling with liquid: Add 6 mL (for 1 mL specification) or 600 μL (for 250 μL specification) of PBS buffer into the column to swell the dry packing material.
2.2 Stabilization: Allow to stand at room temperature for approximately 3 minutes.
2.3 Centrifugation: Centrifuge at 1200×g for 3 minutes, then discard the supernatant. 2.4 Repeat: Add 1 mL (for 1 mL specification) or 200 μL (for 250 μL specification) of the above PBS buffer, and repeat the above balance operation 2 times to ensure that the column is completely balanced in the PBS environment.
3. Sample Processing
3.1 Sample Addition: Add the protein samples to be processed to the center of the balanced column.
Recommended sample volume: 500 - 1000 μL (for 1 mL specification); 150 - 200 μL (for 250 μL specification).
3.2 Static Stabilization: Allow the sample to stand at room temperature for 30 seconds to ensure it fully penetrates the packing bed.
3.3 Centrifugal Collection: Centrifuge at 1200×g for 3 minutes.
3.4 Collection: The liquid collected from the tube at this point is the protein sample after desalination. Please immediately transfer it for storage and future use.
4. Regeneration and Preservation of Columns (Optional)
If you need to reuse the same column, please follow the steps below:
4.1 Scenario A: Use after desalination of protein
4.1.1 Add 1000 μL (1 mL specification) or 200 μL (250 μL specification) of PBS solution containing 2M NaCl a total of 4 times.
4.1.2 A total of 4 times, 1000 μL (1 mL specification) or 200 μL (250 μL specification) of PBS solution containing 2M NaCl was added.
4.1.3 Then, add 1000 μL (in 1 mL specification) or 200 μL (in 250 μL specification) of deionized water three times.
4.1.4 After each addition, let it stand for 2 minutes, and then centrifuge at 1200×g for 3 minutes.
4.2 Scenario B: Washing off small molecule dyes before reuse
Add 20% ethanol solution continuously to the desalination column until the dyes are completely washed away. Then, rinse with deionized water or buffer solution.
4.3 Scene C: Applicable to Different Proteins
4.3.1 Add 1000 μL (1 mL specification) or 200 μL (250 μL specification) of 0.5M NaOH solution.
4.3.2 Let it stand for 2 minutes, then centrifuge at 1200×g for 3 minutes.
4.3.3 Repeat this operation 2 to 4 times.
4.3.4 Then, rinse with deionized water or buffer solution until no NaOH is detected.
5. Storage: After processing, place the desalted column in a 20% ethanol solution and store it.
6. Recycling Effect
The recovery rate of the sample after desalination is related to the concentration of the sample and the volume of the sample added. The higher the sample concentration, the higher the recovery rate; and within the recommended sample addition volume range, the larger the addition volume, the higher the recovery rate will be. Generally, the recovery rate of the protein after desalination is between 80% and 95%.

Notes
(1) The sample processing volume should be within the range of the column processing capacity. Excessive volume will result in incomplete desalination, while insufficient volume will lead to a decrease in sample recovery rate.
(2) The packing may experience water loss and shrinkage when placed in high-concentration alcohol solution or saturated salt solution. Please do not perform column chromatography with these solutions (unless it is a regeneration and cleaning step).
(3) When adding the sample, try to distribute it evenly to the center of the tube to avoid disrupting the smoothness of the column bed surface.
(4) When the sample concentration is too low, it is necessary to first concentrate the sample to the desired volume or concentration, and then perform the desalination process.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.