|Product Overview :||The isolated ER can be used for studying the cytochrome P450 system and xenobiotic metabolism, for studying lipid metabolism, and for the recovery of ER membrane and lumenal proteins. The kit was tested with rat liver, kidney, and brain; mouse liver; rabbit liver, kidney, spleen, heart, and brain; and with Jurkat and HeLa cell lines|
|Applications :||The Endoplasmic Reticulum Isolation Kit contains all the reagents required for ER preparations with various degrees of purity: crude ER (microsomes), Ca2+ precipitated rough ER (RER) enriched microsomes, and density gradient purified rough ER (RER) and smooth ER (SER).|
|Storage :||Store the unopened kit at 2-8 centigrade. When stored unopened, the components in this kit are stable for 24 months. After opening the kit, the needle and the OptiPrep Density Gradient Medium can be stored at room temperature.|
|Kit Components :||Isotonic Extraction Buffer 5× (50 mM HEPES, pH 7.8, with 1.25 M sucrose, 5 mM EGTA, and 125 mM potassium chloride) 100 ml
Hypotonic Extraction Buffer 10× (100 mM HEPES, pH 7.8, with 10 mM EGTA and 250 mM potassium chloride) 10 ml
Calcium Chloride Solution (2.5 M calcium chloride solution) 5 ml
OptiPrep Density Gradient Medium (60% (w/v) solution of iodixanol in water) 100 ml
Needle, 4 inch, 20 gauge 1 each
|Materials Required but Not Supplied :||· Protease Inhibitor Cocktail for use with mammalian
cell and tissue culture extracts
· Dulbecco’s Phosphate Buffered Saline (PBS)
· Ultrapure water
· Sorvall RC-5C centrifuge with SS-34 head or equivalent
· Ultracentrifuge with a Kontron 65Ti fixed-angle
head or equivalent and 8 ml tubes
· Appropriate homogenizer:
- Potter-Elvehjem PTFE pestle in glass tube homogenizer - 3 ml, 8 ml, 45 ml)
- Pellet pestle and Motor for pellet pestle
- Dounce glass tissue grinder set, 7 ml
- Overhead electric motor
· Scalpel, forceps, and glass plate
· Microcentrifuge tubes
· Pasteur pipettes
· Syringe - suitable for gradient separation
(1 ml syringe for an 8 ml ultracentrifuge tube)
|Preparation :||Reagent Preparartion
Note: Use ultrapure water for the reagent preparation.
1. 1× Isotonic Extraction Buffer - 10 mM HEPES, pH 7.8, 250 mM sucrose, 25 mM potassium chloride, and 1 mM EGTA Aseptically remove an aliquot of the Isotonic Extraction Buffer 5× and dilute it 5-fold with water. Keep the 1× Isotonic Extraction Buffer at 4 centigrade before use. Just before use add Protease Inhibitor Cocktail for mammalian cells to the 1× Isotonic Extraction Buffer at a concentration of 1% (v/v).
Note: Suggested volumes of 1×Isotonic Extraction Buffer
· For Option 1 - use a minimal tissue weight of 0.5-1 g and prepare 10 ml of buffer for 0.5–1 g of tissue weight.
· For Option 2 without gradient separation – use a minimal tissue weight of 2 g and prepare 20 ml of buffer for 2 g of tissue weight.
· For Option 2 with gradient separation - use a minimal tissue weight of 4 g and prepare 60 ml of buffer: 40 ml for crude microsome preparation and 20 ml for the density gradient separation (when using two 8 ml centrifuge tubes).
2. 1× Hypotonic Extraction Buffer - 10 mM HEPES, pH 7.8, 25 mM potassium chloride and 1 mM EGTA (required for preparation of ER from cultured cells) Dilute an aliquot of the Hypotonic Extraction Buffer 10× 10-fold with water. Keep the 1× Hypotonic Extraction Buffer at 4 centigrade before use. Just before use add the Protease Inhibitor Cocktail for mammalian cells to the 1×Hypotonic Extraction Buffer at a concentration of 1% (v/v).
Prepare 3 ml of 1× Hypotonic Extraction Buffer for each ml of packed cells.
3. 8 mM Calcium Chloride Solution - Dilute an aliquot of the 2.5 M CaCl2 Solution with water, 0.032 ml of the 2.5 M CaCl2 Solution per 10 ml of water. Keep the 8 mM Calcium Chloride Solution at 4 centigrade before use. Prepare 7.5 ml of the 8 mM Calcium Chloride Solution for each ml of PMF.
4. Optiprep density gradient - For separation in 8 ml centrifuge tubes prepare:
· 10 ml of 30% Optiprep – Dilute the OptiPrep Density Gradient Medium [60% (w/v)] 2-fold with 1× Isotonic Extraction Buffer. Mix well.
· 10 ml of 15% Optiprep – Dilute the OptiPrep Density Gradient Medium [60% (w/v)] 4-fold with 1× Isotonic Extraction Buffer. Mix well.
|Separation Protocol :||A. Preparation of ER from animal tissues
Perform the whole procedure at 4 centigrade. All the solutions and equipment should be pre-cooled before use. For samples below 1 g use an 8 ml PTFE pestle in glass tube homogenizer. For samples greater than 1 g use a 45 ml PTFE pestle in glass tube homogenizer.
1. Use a fresh tissue sample from an animal that was starved overnight and sacrificed the next morning.
2. Wash the tissue sample twice with 10 ml of PBS by placing in a dish and shaking gently for few minutes. Note: This step is performed in order to remove blood from certain tissues (liver). Place the tissue on a paper towel in order to absorb excess liquid and blood clots, if present. Cut the tissue into small pieces (1.5–2 cm) and repeat the wash step.
3. Blot the tissue on a paper towel and weigh.
4. Cut the tissue, with the aid of a scalpel and glass plate, into small slices (0.3–0.5 cm). Transfer the slices into a suitable glass homogenizer. Add 3.5 ml of the 1×Isotonic Extraction Buffer per gram of tissue and homogenize the sample by an overhead motor (~200 rpm). Ensure total homogenization of the sample by moving the pestle up and down at least 7 times. Transfer the homogenate to a centrifuge tube.
5. Wash the PTFE pestle and glass vessel with 0.5 ml of the 1×Isotonic Extraction Buffer per gram of tissue and add to the previous homogenate. Keep the homogenate on ice.
6. Centrifuge the homogenate at 1,000 ´ g for 10 minutes at 4 centigrade. Carefully remove the thin floating lipid layer by aspiration, being careful not to aspirate the post nuclear supernatant. Transfer the supernatant to another centrifuge tube using a pipette and discard the pellet.
7. Centrifuge at 12,000 × g for 15 minutes at 4 centigrade. Carefully remove the thin floating lipid layer by aspiration, being careful not to aspirate the post mitochondrial supernatant. Transfer the supernatant to another tube using a pipette and discard the pellet. This supernatant fraction, which is the post mitochondrial fraction (PMF), is the source for microsomes.
Note: For further analyses it is recommended to save a sample (100-400 ml) before continuing with the next purification step.
Option 1 - For isolation of RER enriched microsomes (precipitation with calcium chloride) Perform this procedure at 4 centigrade.
1. Measure the volume of the PMF (V ml).
2. Prepare a volume of 8 mM Calcium Chloride Solution equivalent to 7.5 times the volume of the PMF (V ml) (instructions in Reagent Preparation section).
3. Transfer the PMF to a beaker (a size of 10 times the volume of the PMF) containing a suitable magnetic spinbar.
4. Add a volume of 8 mM Calcium Chloride Solution equivalent to 7.5 times the volume of the PMF, dropwise, to the PMF with constant stirring (a burette, Pasteur pipette, or separatory funnel may be used for dropwise addition of the solution). The final concentration of CaCl2 is 7 mM.
5. After all the 8 mM Calcium Chloride Solution is mixed with the PMF, stir for additional 15 minutes at 4 centigrade.
6. Centrifuge the sample at 8,000 × g for 10 minutes at 4 centigrade. The enriched RER microsomes will be in the pellet.
7. Remove the supernatant and suspend the pellet in 1× Isotonic Extraction Buffer (0.3 ml of buffer for each g of original tissue).
8. Place the suspension in a homogenizer. If the volume of the suspension is small (less than 0.8 ml) use a pellet pestle in a microcentrifuge tube. If the volume is larger than 0.8 ml, use a 3 ml PTFE pestle in glass tube homogenizer.
9. Homogenize completely by moving the pestle up and down several times at ~200 rpm. Option 2 - For isolation of the crude microsomal fraction (ultracentrifugation)
Perform this procedure at 4 centigrade.
1. Centrifuge the PMF for 60 minutes at 100,000× g in an ultracentrifuge at 4 centigrade. The PMF may be divided into two or more tubes. The pellet is the microsomal fraction.
2. Remove the supernatant using a pipette and discard.
3. The pellet is difficult to suspend. Therefore, transfer it to an appropriate homogenizer with the aid of a spatula.
4. Wash each ultracentrifuge tube with 1× Isotonic Extraction Buffer (0.3 ml for each g of original tissue) and transfer the liquid to the homogenizer vessel. If the volume of the suspension is small (less than 0.8 ml) use a pellet pestle in a microcentrifuge tube. If the volume is larger than 0.8 ml, use a 3 ml PTFE pestle in glass tube homogenizer.
5. Homogenize completely by moving the pestle up and down several times at ~200 rpm. Notes: In some tissues (brain) the ER does not pellet well and only 25% of the ER will be in this pellet. Liver tissue, on the other hand, will give 70–90 % of the ER in the pellet.
For further analyses it is recommended to save a sample (100–400 ml) before continuing with the next purification step.
Self-Generating Density Gradient (OptiPrep) This procedure is for further purification and separation of RER (rough endoplasmic reticulum) and SER (smooth endoplasmic reticulum) from microsomes isolated by ultracentrifugation. This procedure is based on the use of the selfgenerating density gradient medium iodixanol (OptiPrep). Iodixanol is a low osmolarity, iodinated density gradient medium that is biologically inert and does not interfere with assays for marker enzymes. The crude microsomal fraction is adjusted to 20% (w/v) Optiprep and is layered between 30% and 15% Optiprep layers. Following ultracentrifugation using a fixed angle rotor, fractions are separated from the top to the bottom of the gradient.
This procedure is suitable for 1–2 ml of the crude microsomal suspension (100,000 ´ g pellet), equivalent to 3.5–7 g of tissue or approximately 1–5 x 109 cells, when centrifuged in 8 ml ultracentrifuge tubes. For larger amounts adjust the procedure accordingly.
Prepare a calibration scale sheet that suits the ultracentrifuge tubes used, in order to facilitate the sample removal:
1. Affix a plain piece of paper or preferably a small transparent sheet of plastic to the outside of the ultracentrifuge tube to be used.
2. Add aliquots of water equal to the volume of the desired fractions (0.5 ml) to the tube and mark the sheet with a fine tipped marker at the height of the liquid. Use this as a guide for removal of identical aliquots after centrifugation.
Perform this procedure at 4centigrade
1. Dilute the crude microsomal sample (Procedure A, Option 2, step 5) with the 60% OptiPrep Density Gradient Medium to a final concentration of 20% Optiprep (Add 0.5 ml of the 60% OptiPrep Density Gradient Medium per 1 ml of sample). Mix well.
2. Take an 8 ml ultracentrifuge tube and place 2 ml of the 30% Optiprep solution at the bottom.
3. Carefully layer the sample containing 20% Optiprep (step 1), up to 1.5 ml, on top of the 30% layer with the aid of a Pasteur pipette, by placing drops on the wall of the tube close to the bottom layer. Ensure that the entire sample floats on top of the 30% Optiprep solution cushion.
4. Layer 4 ml of the 15% Optiprep solution very carefully on top of the sample with the aid of a Pasteur pipette, as described in step 3.
5. Close the tube and ensure that the balance tube is at the same weight as the sample tube (a similar gradient or another sample may be used as balance).
6. Centrifuge in an ultracentrifuge for 3 hours at 150,000× g.
7. At the end of the run carefully remove the tube and clamp it to a stable base.
8. Affix the calibration scale sheet to the side of the tube with tape such that the upper meniscus coincides with a calibration line.
9. For an 8 ml centrifugation tube, withdraw fractions of 0.5 ml from the top of the gradient downwards using the supplied 4-inch blunt ended needle and a suitably sized syringe (insert the end of the needle to the bottom of the first calibration line and withdraw the aliquot). Record the volume withdrawn, as there may be variations between samples. Transfer the fraction to a microcentrifuge tube and close the tube.
10. Continue to withdraw fractions in the same fashion by moving the needle to the bottom of the next calibration line. Transfer each fraction to a new microcentrifuge tube and close the tube.
11. After removing the supernatant, wash the tube with 200 ml of 1×Isotonic Extraction Buffer to suspend any pellet.
12. Label the tubes containing the samples and perform appropriate assays. It may be useful to determine the following parameters: Protein concentration with Bradford’s reagent (10-fold dilution, 10–30 ml sample per test) NADPH cytochrome c reductase activity (ER marker) - use 5–20 ml sample per test. Use the Cytochrome c Reductase (NADPH) Assay Kit. RNA detection (10–50 ml sample per test) for the identification of RER, to which ribosomes are attached, in contrast to SER which should be devoid of RNA.
B. Preparation of microsomes from cultured cells Microsomes may also be prepared from tissue culture cells using the above procedure except for the initial extraction procedure (Procedure A, steps 1-7), which may have to be altered as follows: For adherent cells (such as HeLa cells, >2 x 10^8 cells):
1. Detach cells using conventional tissue culture methods.
2. Centrifuge the cells at 600×g for 5 minutes and remove the supernatant by aspiration.
3. Wash the cells with 10 volumes of PBS and centrifuge as in step 2.
4. Measure the packed cell volume (PCV). 5. Suspend the cells in a volume of 1×Hypotonic Extraction Buffer equivalent to 3 times the PCV and incubate the cells for 20 minutes at 4 centigrade to allow the cells to swell.
6. Centrifuge the cells at 600×g for 5 minutes and remove the supernatant by aspiration. Measure the “new” PCV.
7. Add a volume of 1´ Isotonic Extraction Buffer equivalent to 2 times the “new” PCV and transfer to a 7 ml Dounce homogenizer.
8. Break the cells with 10 strokes of the Dounce homogenizer and then proceed to the differential centrifugation steps (Procedure A, steps 6-7). For fragile cells (with “weak” outer membranes such as Jurkat cells):
1. Perform steps 2-4 of the procedure for adherent cells.
2. Suspend the cells in a volume of 1×Isotonic Extraction Buffer equivalent to 4 times the PCV.
3. Homogenize the cells using a Dounce homogenizer or by passing the cells through a 27
4. Proceed to the differential centrifugation steps (Procedure A, steps 6-7).