The goal of the following protocol is to generate subcellular fractions from frozen or fresh gastrointestinal specimens. Proteins take part in almost all of your biological functions within a cell. In any variation they are extracted amongst a location can lead to a pathogenic scenario.
Some studies will take advantage of the assessment of protein localization or will need to invade a protein from a specific cellular compartment. So the reproducible fractionation of intact proteins is important in this respect. Intestinal biopsy samples are recently obtainable during endoscopy procedures and can be effectively used for protein quantification or immuno precipitation studies.
Clinical intestinal samples are an important resource for the effective identification of tissue and disease specific proteins. The name of this method is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of a specific protein or protein complexes. This method serves for the fractionation of both.
First, our frozen intestinal biosis happens. For the frozen tissue procedure, freeze samples immediately after extraction and store in liquid nitrogen until use. It is important to maintain the sample frozen until the cold homogenization buffer is added.
For fresh samples, transfer the biopsy to a 1.5 mL microtube and keep on ice until the procedure is started. Frozen tissue samples are usually attached to the wall of the cryotube, the tissue can be released using a sterile pipette tip before transferring to a conical 1.5 mL microtube. Add 200 mL of buffer one supplemented with DDT and phosphatase and protease inhibitors to the microtube containing the sample.
Homogenize the sample with a vessel until the tissue is completely disrupted and keep it on ice. Incubate the sample on ice for 10 minutes. Add 10 mL of 1 percent detergent to each sample to a final concentration of 0.05 percent in order to disrupt the cell membrane.
Incubate on ice for five minutes. Vortex briefly and centrifuge at 400 g at 4 degrees for two minutes. Remove the supernatant containing the cytoplasmic protein fraction and transfer it to a clean tube.
Do not discard the pellet. Re suspend the pellet in 200 mL of buffer two supplemented with DDT and phosphatase and protease inhibitors. Centrifuge at 400 g for 2 minutes.
Discard the supernatant and repeat the washing procedure two more times. After the third wash, re suspend the pellet in 100 mL of buffer two. Shake vigorously at 4 degrees for 30 minutes.
Centrifuge at 4 degrees at top speed for 10 minutes. Transfer the supernatant containing the nuclear protein fraction to a clean tube. Cytoplasmic and nuclear fractions are now ready for quantification and downstream experiments.
The intestinal mucosa is rich in several proteases and therefore protein degradation has to be controlled during extraction in order to minimize protein degradation, multiple protease inhibitors should be included in the protein extraction buffers. Additionally, the extracts are to be used for additional assays. It is essential to avoid protein denaturation or proteolysis.
As this cause a loss of protein activity. As a consequence of the cell-cell and cell-matrix contacts that are present in tissues, some modifications have to be implemented in the protein extraction protocols routinely used with cell cultures. Separation of the cells from the transcellular matrix without affecting protein quality is a critical factor for the efficient isolation of intestinal tissue proteins.
To the left, the table shows the protein concentrations of each of the fractions from the fresh and frozen 2 mL biopsy samples. Concentrations are shown in microgram per microliter. In this experiment, a fresh and frozen sample were simultaneously fractionated following the protocol.
And a Western blot was performed using 20 mg of each sample. To check the purity of the fractions, HSP90 and Tubulin were used as cytoplasmic controls and HDAC1 and H3 as nuclear controls. As can be observed, HSP90 and Tubulin are primarily located in the cytoplasm and HDAC1 and H3 in the nucleus with very residual mixing between the two fractions.
To see whether the protocol affects cell death, we also detected CASP3. Both samples present a very minimal staining for CASP3 confirming that the procedure does not affect cell death pathway even after flash-freezing the biopsies. The frozen samples used in this experiment came from an individual with an intestinal inflammatory condition and as expected, pSTAT1 appears in the cytoplasm of this sample, confirming the protein modifications are maintained after fractionation, and thus can be used for downstream functional analysis.
Reproducibility of the results in frozen biopsies is shown in this figure. Fractions are clean with minimal compartment protein mixing. Fresh and frozen biopsies can be equally used in these protocols, with frozen biopsies giving clean and reproducible results as seen in Figure 1 and 2.
The protocol described here is designed to generate the possible protein fractions. However, some intrinsic variability within the sample including the size and status of the tissue can lead to deviations in the protein amounts and the distribution amounts in the fractions.
