Methodology for the Extraction of Proteins from Serum. Learning Objectives: Proteomics Extraction of Proteins from Serum

Transcription

1 Proteomics Extraction of Proteins from Serum Methodology for the Extraction of Proteins from Serum Extraction of the entire protein from the complex biological samples like serum requires a multi -step robust protocol, where various sample processing steps have been introduced to increase efficacy of the extraction procedure. Learning Objectives: After interacting with this learning object, the learner will be able to: Define the protein precipitation using TCA-Acetone treatment. Infer the protein solubilisation using rehydration buffer. Separate out high abundance proteins. Operate steps involved in handling the instrument and the material used. Assess the troubleshooting steps involved in the experiment. Note: The current IDD exists in two modes- interactive and automatic. Students taking lab course should select interactive (set as default), while the automatic mode may be selected for general users.

2 Reagents Preparation Clean the surface of the balance and tare the weight of paper before weighing the reagents.

3 Reagents Preparation Prepare 10% TCA-Acetone solution by dissolving 1 gram of trichloroacetic acid and 0.07 grams of Dithiotreitol in 10 ml of acetone. Dissolve completely by vortexing the tube and store the tube at 4 C for later use.

4 Reagents Preparation Weigh 0.02 grams of CHAPS, 0.6 grams of urea and dissolve in water to prepare rehydration buffer. Add 500μl of bromophenol blue to the tube, vortex it thoroughly and store the tube at 4 C for later use.

5 Blood Processing For serum sample, collect blood from a donor in a serum separation tube. This should be done very carefully under the supervision of a skilled person to avoid any accidents.

6 Blood Processing Place the tube on ice for about an hour and allow coagulation of the collected blood sample. After incubation, centrifuge the tube at 2500 rpm, 4 C for 10 minutes to separate serum from the coagulated blood.

7 Blood Processing Collect the upper layer of straw colored serum into a fresh eppendorf tube for further processing.

8 Blood Processing Make aliquots of serum by distributing into fresh eppendorf tubes and store the tubes at -80 C. The tubes can be used later when required.

9 Sample Processing Take out the required amount of serum into a fresh eppendorf tube from the serum stock maintained at -80 C. Place the serum stock tube back in the -80 C freezer after use.

10 Sample Processing Add 500μl of Phosphate buffer to the serum sample taken in a fresh eppendorf tube.

11 Sample Processing Vortex the sample tube to bring about uniform mixing of serum with phosphate buffer. Proceed for cell lysis by sonication once the mixing is complete.

12 Sample Processing Sonicate the sample for 8 cycles, 20% amplitude 5 seconds on and 15 seconds off. The step is to be carried out by placing on ice since a lot of heat is released during sonication.

13 Sample Depletion Serum contains a large number of high molecular weight proteins like Albumin, α1-antitrypsin, Transferrin, Haptoglobulin, Immunoglobulin G and Immunoglobulin A. These proteins interfere with the separation of low molecular weight proteins during IEF and hence should be depleted from serum.

14 Sample Depletion Take the depletion column out of the refrigerator in order to ready it for pretreatment.

15 Sample Depletion Once the column attains the room temperature centrifuge it at 800rpm for 5minutes. This separates the storage buffer from the resin.

16 Sample Depletion Remove binding buffer from the freezer and add it to the column. Centrifuge the column at 2400rpm for 5minutes. The binding buffer helps in activating the resin thereby causing better binding.

17 Sample Depletion Discard the buffer in the column. The column is now ready to be used. Add serum to the column and incubate it on ice for 10 minutes.

18 Depletion Mechanism The proteins of high abundance bind to the resin while those of low abundance pass through the column. The depleted sample is now better suited for separation using 2D gel electrophoresis.

19 TCA-Acetone Precipitation Centrifuge the column for 30seconds at 800 rpm. This helps in better separation of the high and low abundance proteins.

20 TCA-Acetone Precipitation Collect the depleted sample in a separate tube. Add 500 μl of 10% TCA-Acetone solution to the tube and place it in the -20 C freezer for 4 hours to precipitate the proteins.

21 TCA-Acetone Precipitation Centrifuge the sample at rpm, 4 C and 30 minutes. The proteins would be seen as a pellet at the bottom of the tube.

22 TCA-Acetone Precipitation Separate the supernatant from the pellet by careful pipetting. Take care not to disturb the pellet.

23 TCA-Acetone Precipitation Add 1ml of ice cold wash buffer for pellet wash.

24 TCA-Acetone Precipitation Disperse the pellet in the wash buffer that has added by adequate vortexing.

25 TCA-Acetone Precipitation Now centrifuge the tube at 14000rpm, 4 C for 10 minutes to pellet the proteins.

26 Rehydration Buffer Treatment Take the tube out of the centrifuge and discard the supernatant. Air-dry the pellet for 10 minutes and add 400μl of rehydration buffer, sufficient to dissolve the pellet. Vortex the tube to dissolve pellet completely. Rehydration buffer consists of urea which denatures the proteins and CHAPS which solubilizes proteins.

27 Sample Storage Store the sample in -20 C freezer until further use.

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