Product Introduction:
This kit is used to enrich low-abundance proteins in plasma samples and rapidly prepare proteomic samples, and the processed samples can be used for subsequent mass spectrometry detection.
Product Components and Storage Conditions:
M1456374
| Component
| 10T
| 25T
| Storage
|
M1456374 A
| Magnetic beads Max
| 200 μL
| 500 μL
| 4℃
|
M1456374 B
| Incubation Buffer I
| 4.8 mL
| 12 mL
| RT
|
M1456374 C
| Incubation Buffer II
| 6 mL
| 15 mL
| RT
|
M1456374 D
| Washing Buffer
| 6 mL
| 15 mL
| RT
|
M1456374 E
| Lysis Buffer
| 0.6 mL
| 1.5 mL
| -20℃. Store in the dark.
|
M1456374 F
| Balance Buffer
| 2.8mL
| 7 mL
| 4℃
|
M1456374 G
| Digest Buffer | 16 μL
| 40 μL
| -20℃
|
M1456374 H
| Stop Buffer
| 300 μL
| 750 μL
| RT
|
M1456374 I
| Wash Buffer I
| 4 mL
| 10 mL
| RT
|
M1456374 J
| Wash Buffer II
| 2.4 mL
| 6 mL
| RT
|
M1456374 K
| Wash Buffer III
| 2.4 mL
| 6 mL
| RT
|
M1456374 L
| Elution Buffer
| 4.8 mL
| 12 mL
| RT. Store in the dark.
|
M1456374 M
| Loading Buffer
| 120 μL
| 300 μL
| RT
|
M1456374 N
| Tip pillar
| 10T
| 25T
| RT |
Operating Procedure:
1.Centrifuge the plasma sample (3000g, 10min), and take the supernatant for later use (if storage is required, store it at -80°C for long-term preservation to avoid repeated freezing and thawing).
2.Take 50-100μL of centrifuged plasma, add 400μL of Incubation buffer I, then add 18μL of Magnetic beads, vortex to mix, and incubate at room temperature on a shaking mixer for 1 hour.
3.After incubation, use a magnetic rack to magnetically separate for 3min, and discard the supernatant.
4.Remove the EP tube, add 500μL of Incubation buffer II, gently invert up and down to mix several times, magnetically separate for 3min, and discard the supernatant.
5.Remove the EP tube, add 500μL of Washing Buffer, gently invert up and down to mix several times, magnetically separate for 3min, and discard the supernatant.
6.Remove the EP tube, add 50μL of Lysis Buffer to resuspend the beads, place in a water bath at 95°C for 10min, then take it out and cool to room temperature.
7.Add 225μL of Balance buffer to the EP tube that has cooled to room temperature.
8.Add 1μL of Digest buffer to the sample, and perform enzymatic digestion with shaking in a metal bath at 37°C and 1200rpm for 3-16h.
9.After enzymatic digestion, take out the EP tube, add 25μL of Stop buffer to the sample, and vortex to mix.
10.Add 320μL of Wash buffer I, shake vigorously for 3min, centrifuge at 15000rpm for 3min, and remove the upper liquid.
11.Transfer the lower layer sample into the Tip column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down. If the liquid flow rate is slow, the rotation speed can be appropriately increased.
12.Add 200μL of Wash buffer II (shake for 10-20s before use) to the desalting column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down.
13.Add 200μL of Wash buffer III to the desalting column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down.
14.Put the desalting column into a new EP tube, add 200μL of Elution buffer to the desalting column, centrifuge at 2000rpm for 3-5min until all the liquid is centrifuged down.
15.Repeat the previous step, collect the eluates from both times, and freeze-dry them.
16.Add 10μL of Loading buffer, vortex vigorously for 3min, centrifuge at 2000g for 10min, take an appropriate amount of sample for mass spectrometry detection. Taking the HF-X instrument as an example, 0.5-1μg of sample is sufficient for loading.
Automated Operation Process:
It is compatible with mass spectrometry proteomics pretreatment workstations, enabling one-stop processing from plasma to peptides. This eliminates manual operation errors, improves the reproducibility of sample preparation workflows, and provides high precision and reliability for various laboratory procedures.
ZJ25F0926873