ab139409 Membrane Fractionation Kit Instructions for Use For the rapid and simple separation of membrane, cytosolic and nuclear cellular fractions. This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Introduction.3 2. Assay Summary.4 3. Kit Contents 5 4. Storage and Handling 5 5. Additional Materials Required..5 6. Preparation of Reagents...6 7. Sample Preparation...7 8. Performance and Specificity.9 9. Frequently Asked Questions..12 10. Troubleshooting 14 2
1. Introduction Principle: ab139409 provides the method and reagents for rapid separation of cytosolic, membrane and nuclear fractions from a wide variety of cell culture lines and species of origin. The kit is based on a sequential detergent-extraction of cytosolic and membrane proteins into the extracellular buffer using proprietary detergents. This method circumvents the need for time consuming and inefficient cell mechanical disruption and differential centrifugation steps. The ab139409 kit fractionates the cells into cytosol-containing, membrane-containing and nuclei-containing fractions. These fractions are referred throughout the protocol as cytosolic, membrane and nuclear fractions. In the first step, the plasma membrane is selectively permeabilized with Detergent I. The cytosol-containing fraction is separated from the remaining cellular material containing membranes and nuclei by a simple centrifugation step. In the second step, membrane proteins are then extracted with Detergent II and separated from the nucleicontaining fraction by a second centrifugation step. The three distinct fractions generated can be run by gel electrophoresis and analyzed by Western Blot or run on high-performance liquid chromatography and analyzed by mass spectrometry. 3
2. Assay Summary Harvest cells in their own media and wash once in 1X Buffer A. Resuspend the cell pellet in 1X Buffer A, mix with an equal volume of Buffer B and incubate. Cytosolic Fraction = Centrifuge sequentially at 5,000 x g for 1 min at 4 C (A1), followed by centrifugation of A1 supernatant at 10,000 x g for 1 min 4 C (B1) Combine pellets A1 and B1 in 1X Buffer A to the same volume of the original cell suspension, mix with an equal volume of Buffer C and incubate Membrane Fraction = Centrifuge sequentially at 5,000 x g for 1 min at 4 C (A2) followed by centrifugation of A2 supernatant at 10,000 x g for 1 min 4 C (B2) Nuclear Fraction = Combine pellets A2 and B2 in 1X Buffer A to the same volume of the original cell suspension. 4
3. Kit Contents Sufficient materials are provided for fractionation of 1 x 10 8 cells or for preparation of 40 samples, each corresponding to one 100 mm plate at 2.5 x 10 6 cells / plate. 2X Buffer A 175 ml Detergent I 25 µl Detergent II 5X SDS Sample Buffer 1 ml 10 ml 4. Storage and Handling Store 2X Buffer A, Detergent II and 5X SDS Sample Buffer at -20 C, store Detergent I at -80 C. 5. Additional Materials Required Centrifuge capable of at least 10,000 x g 1.5 ml microfuge tubes. Cell counting device such as hematocytometer. Protease inhibitor Cocktail (optional) 5
6. Preparation of Reagents 6.1 Prepare 1X Buffer A by first equilibrating 2X Buffer A to room temperature (RT). Dilute 2X Buffer A with an equal volume of ultrapure water and mix gently. Note: Optionally, 1X Buffer A can be supplemented with a protease inhibitor cocktail to minimize nonspecific proteolysis during the fractionation. 6.2 Immediately prior to use, prepare Buffer B by diluting Detergent I 1000-fold in 1X Buffer A (i.e. 5 ml of 1X Buffer A + 5 µl of Detergent I). Mix well by pipetting. Discard any excess at the end of the experiment. 6.3 Immediately prior to use prepare Buffer C by diluting Detergent II 25-fold in 1X Buffer A (i.e. 4.8 ml of 1X Buffer A + 0.2 ml of Detergent II). Mix well by pipetting. Discard any excess at the end of the experiment. 6
7. Sample Preparation Note: The protocol contains detailed steps for preparation of fractions and analysis by western blot. Do not deviate from the specified protocol steps or optimal results may not be obtained. 7.1 Grow cells in tissue culture dishes or flasks with the desired treatments or experimental conditions including a control. 7.2 Harvest cells according to standard cell culture protocols and centrifuge 300 x g for 5 min at RT. 7.3 Wash the cell pellet once in 5 ml of 1X Buffer A and count on a standard Hemacytometer. Centrifuge 300 x g for 5 min at RT. 7.4 Discard the supernatant and re-suspend cells in 1X Buffer A according to the table below. Cell Line Concentration HeLa 6.6 x 10 6 / ml 143B Osteosarcoma 6.6 x 10 6 / ml HepG2 6.6 x 10 6 / ml Fibroblasts (HdFn) 6.6 x 10 6 / ml SHSY5Y 6.6 x 10 6 / ml Jurkat 2 x 10 7 / ml 7.5 Transfer the cell suspension into a new tube. Add an equal volume of Buffer B to the cell suspension and mix by pipetting. 7
7.6 Incubate samples for 7 minutes on a rotator at RT. 7.7 Centrifuge samples 5,000 x g for 1 min at 4 C. Carefully remove the supernatant and transfer it to a new tube. Save the pellet on ice. 7.8 Centrifuge the supernatant obtained on step 7.7 at 10,000 x g for 1 min at 4 C. Carefully remove the supernatant and transfer it to a new tube labeled Cytosolic Fraction. Save the pellet on ice. 7.9 Resuspend pellets obtained on 7.7 and 7.8 in the same volume of 1X Buffer A used in step 7.4. 7.10 Transfer the volume to a new tube. Add an equal volume of Buffer C to the suspension and mix by pipetting. 7.11 Incubate for 10 minutes on a rotator at RT. 7.12 Centrifuge at 5,000 x g for 1 min at 4 C. Carefully remove the supernatant and transfer it to a new tube. Save the pellet on ice. 7.13 Centrifuge the supernatant obtained on step 7.12 at 10,000 x g for 1 min at 4 C. Carefully remove the supernatant and transfer it to a new tube labeled Membrane Fraction. Save the pellet on ice. 7.14 Resuspend pellets obtained on 7.12 and 7.13 in the same volume of 1X Buffer A used in step 7.4. 7.15 Transfer these samples (7.14) to a new set of tubes labeled Nuclear Fraction. 7.16 For western blot analysis, mix four volumes of sample with one volume of 5X SDS-PAGE Sample Buffer. 8
7.17 Samples may be flash frozen at -80 C before or after the addition of 5X SDS-PAGE Sample Buffer. 8. Performance and Specificity The performance of this kit was tested by running 20 µl of HeLa whole cell lysates and 20 µl of cell fractions lysates on a 4-15% gradient polyacrylamide gel. Membranes were blocked in 5% milk in Tris-buffered Saline containing 0.1% Tween-20 (TBST) for 1 hour and then blotted with primary antibodies against specific organelle protein markers or antibody cocktails overnight at 4 C, followed by species specific secondary antibodies for 2 hours at RT. All antibodies were diluted in 5% Milk-TBST. Figure 1. Analysis of Fractions by Western Blot with a Lysosome Marker. Samples were loaded as follows from left to right: (1) Marker, (2) whole cell lysate, (3) cytosolic fraction lysate, (4) membrane fraction lysate and (4) nuclear fraction lysate. Membrane was blotted with ab25631 anti-lamp2 antibody at 1/5000 dilution 9
Figure 2. Analysis of Fractions by Western Blot with an Endoplasmic Reticulum Marker. Samples were loaded as follows from left to right: (1) Marker, (2) whole cell lysate, (3) cytosolic fraction lysate, (4) membrane fraction lysate and (5) nuclear fraction lysate. Membrane was blotted with ab5484 anti- PDI antibody at 1/1000 dilution Figure 3. Analysis of Fractions by Western Blot with a Mitochondria antibody cocktail. Samples were loaded as follows from left to right: (1) Marker, (2) whole cell lysate, (3) cytosolic fraction lysate, (4) membrane fraction lysate and (5) nuclear fraction lysate. Membrane was blotted with a Mitochondria Fractionation WB cocktail ab139416 containing anti-atp5a antibody (55 kda), anti-gapdh antibody (37 kda) and anti-histone H3 (di methyl K9) antibody 10
Figure 4. Analysis of Fractions by Western Blot with a Plasma membrane antibody cocktail. Samples were loaded as follows from left to right: (1) Marker, (2) whole cell lysate, (3) cytosolic fraction lysate, (4) membrane fraction lysate and (5) nuclear fraction lysate. Membrane was blotted with a Plasma Membrane Fractionation WB cocktail ab139409 containing anti-sodium Potassium ATPase antibody (110 kda), anti-gapdh antibody (37 kda) and anti-histone H3 (di methyl K9) antibody. Figure 5. Analysis of Fractions by Western Blot with ER antibody cocktail. Samples were loaded as follows from left to right: (1) Marker, (2) whole cell lysate, (3) cytosolic fraction lysate, (4) membrane fraction lysate and (5) nuclear fraction lysate. Membrane was blotted with Endoplasmic Reticulum Fractionation WB cocktail ab139415 containing anti-grp78 antibody (78 kda), anti-gapdh antibody (37 kda) and anti-histone H3 (di methyl K9) antibody. 11
9. Frequently Asked Questions 9.1 1X Buffer A has a precipitate, can I still use it? When 2X Buffer A is thawed and 1X Buffer A is prepared, the formation of white precipitate is normal. To dissolve the precipitate, incubate the samples 10 min in a hot water bath with occasional inversion. 9.2 I want to fractionate a cell line other than the ones specified in the protocol? The appropriate permeabilization conditions depend on the ratio of detergent to total cellular mass. As a rule of thumb large cells (like HeLa) should be resuspended at 6.6 x 10 6 cells / ml whereas small cells (like jurkat) should be resuspended at 2 x 10 7 cells / ml. For other cell types, we recommend user determine the optimal ratio of Detergent I to the total cellular protein empirically. This can be accomplished by preparing a two-fold dilution set of cell suspensions in 1X Buffer A. To each sample, add equal volume of Buffer B and follow the steps in the protocol. The optimal condition is the lowest detergent to protein ratio in which GAPDH signal is present in the cytosolic fraction and absent in the corresponding membrane fraction. 9.3 At which point in the protocol can I measure protein concentration in the fractions? 12
A protein assay is optional during the preparation of cell fractions. This can be accomplished by saving a small aliquot of the cell suspension from step 7.3 for subsequent determination of total protein in the whole cell suspension. This aliquot may be solubilized with a detergent of choice and protein measured with a protein assay of choice. Subsequently, when fractions are analyzed, load fractions derived from control and experimental cells in amounts proportional, respectively, to the equal amount of protein in the whole cell suspension. 9.4 Can I use the fractions obtained with this kit for applications other than Western blot? Yes, the detergents used in this kit are compatible with standard ELISA assays as long as the 5X SDS-PAGE Sample Buffer is not added to the fractions. Similarly, these fractions can also be processed by HPLC for further analysis by mass spectrometry. 13
10. Troubleshooting Problem Cause Solution Not enough sample loaded Use gels with wider wells to load more sample per well Inaccurate cell counting Dilute or concentrate the cells to within the range of standard automated counters of hemacytometers No signal by western blot Loss of cells due to detachment during experimental conditions Collect the cells that detached during the experimental conditions and mix with cells obtained during trypsinization of cells. Western blot protocol issues Use one of the antibody or antibody cocktails featured in this product to control for the fractionation procedure. 14
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