Product Introduction:
At present, the conventional proteome sample pretreatment process has many problems, such as poor reproducibility, poor method stability, low sensitivity, being very time-consuming, and inability to be automated. To address the above issues, Aladdin has launched a new generation of "All In One Tube" innovative products. This product is mainly designed for the processing of trace proteins (5-100μg). Even researchers without omics background can quickly prepare pretreated samples, which can be used for subsequent mass spectrometry analysis.
Experimental Flowchart of Proteomics Pretreatment Kit (P1456469)
Product Components and Storage Conditions:
P1456469
| Component
| 12 T
| 24T
| 48T
| Storage
|
P1456469A
| Lysis Buffer
| 1.25 mL
| 2.5 mL
| 5 mL
| -20℃. Store in the dark. |
P1456469B
| Digest
| 0.05 mL
| 0.1 mL
| 0.2 mL
| -20℃
|
P1456469C
| Balance Buffer
| 3 mL
| 6 mL
| 12 mL
| 2-8℃
|
P1456469D
| Stop Buffer
| 0.375 mL
| 0.75 mL
| 1.5 mL
| RT |
P1456469E
| Wash Buffer I
| 5 mL
| 10 mL
| 20 mL
| RT
|
P1456469F
| Wash Buffer II | 3.75 mL
| 7.5 mL
| 15 mL
| RT
|
P1456469G
| Wash Buffer III
| 3.75 mL
| 7.5 mL
| 15 mL
| RT
|
P1456469H
| Elution Buffer
| 6.25 mL
| 12.5 mL
| 25 mL
| RT. Store in the dark. |
P1456469I
| Loading Buffer
| 0.25 mL
| 0.5 mL
| 1 mL
| RT
|
P1456469J
| Tip pillar
| 12 T
| 24T
| 48T
| RT |
Trace proteins: Designed for the pretreatment of 5-100μg proteins.
Simple operation: Enables rapid preparation of pretreated samples, requiring only a metal bath and a conventional centrifuge.
High stability: Strict quality inspection for each batch ensures high reproducibility of experimental results.
Operating Procedure:
Lysis
① Add 50μL of Lysis buffer to the EP tube containing the sample and mix by shaking.
② Place the EP tube with Lysis buffer in a 95°C water bath for 10 minutes, then take it out and cool to room temperature.
Enzymatic Digestion
① Add 225μL of Balance buffer to the EP tube that has cooled to room temperature.
② Add 3μL of Digest, mix well, and perform enzymatic digestion with shaking at 37°C and 1200rpm overnight (16 hours is recommended).
Desalting (at room temperature)
① Add 25μL of Stop buffer to the sample and vortex to mix.
② Add 320μL of Wash buffer 1, shake vigorously for 3 minutes, centrifuge at 15000rpm for 3 minutes, and remove the supernatant.
③ Transfer the lower layer sample into the Tip column, centrifuge at 2500rpm for 3-5 minutes until all liquid is centrifuged down. If the liquid flow rate is slow, the speed can be increased.
④ Add 200μL of Wash buffer 2 (shake for 10-20 seconds before use) to the desalting column, centrifuge at 2500rpm for 3-5 minutes until all liquid is centrifuged down.
⑤ Add 200μL of Wash buffer 3 to the desalting column, centrifuge at 2500rpm for 3-5 minutes until all liquid is centrifuged down.
⑥ Put the desalting column into a new EP tube, add 200μL of Elution buffer to the desalting column, centrifuge at 2000rpm for 3-5 minutes until all liquid is centrifuged down.
⑦ Repeat step ⑥, collect the eluates from both times, and freeze-dry them.
⑧ Add 10μL of Loading buffer, vortex vigorously for 3 minutes, centrifuge at 20000g for 10 minutes, take an appropriate amount of sample, and then mass spectrometry detection can be performed. Taking the HF-X instrument as an example, 0.5-1μg of sample is sufficient for loading.
Precautions:
After aliquoting, Digest should be stored at -20°C.
After aliquoting, Lysis Buffer should be stored at -20°C and avoid repeated freezing and thawing.
This product is limited to scientific research use by professionals, and must not be used for clinical diagnosis or treatment, nor for food or drugs.
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