Cell Membrane and Cytoplasmic Protein Extraction Kit, IC-7910

Product Introduction

This kit is designed for the separation of cytoplasmic and membrane proteins from cultured cells and tissue samples. The principle is as follows: cells or tissues are first gently pretreated with Lysis Buffer, which promotes cell lysis and weakens cellular structures while exhibiting low solubilizing capacity for membrane proteins and minimal disruption of membrane structure. Cells are then disrupted by homogenization, and nuclei and unbroken cells are removed by low-speed centrifugation. Membrane fragments are subsequently pelleted by high-speed centrifugation, with the supernatant representing the cytoplasmic protein fraction. Finally, membrane proteins are solubilized from the membrane pellet using Membrane Protein Solubilization Buffer, and the supernatant collected after centrifugation represents the membrane protein fraction. This kit is suitable for Western blot, immunoprecipitation, and other protein analysis applications. For research use only; not for diagnostic or therapeutic procedures.

Product Features

1. Stepwise Lysis and Membrane Protein Solubilization: Lysis Buffer promotes cell lysis, while Membrane Protein Solubilization Buffer is specifically used for membrane protein solubilization, reducing premature release of membrane proteins.

2. Gentle Pretreatment: Lysis Buffer exhibits weak disruption of membrane structure, facilitating subsequent separation of membrane and cytoplasmic fractions.

3. Controllable Operation: Provides specific operating parameters including microscopic criteria for cell disruption, leaving residual liquid when aspirating supernatant, and vortex extraction with Membrane Protein Solubilization Buffer.

4. Broad Sample Compatibility: Cultured cells (adherent/suspension) and tissue samples.

5. Quality Control Guidance: Provides validation protocols using membrane/cytoplasmic markers to assess fraction purity.

6. Compatible with Phosphoprotein Analysis: Phosphatase inhibitors can be added as needed to protect phosphorylation modifications.

7. 5¡Á Loading Buffer Included: Facilitates downstream Western blot sample preparation; heat-sensitive membrane proteins may be gently denatured at 37¡ãC for 30 minutes.

Kit Components and Functional Roles

1. Lysis Buffer

Function: Cell lysis/pretreatment buffer; promotes cell lysis with low solubilizing capacity for membrane proteins and weak disruption of membrane structure.

Stage of Use: Pretreatment prior to cell/tissue homogenization.

Note: Add protease inhibitor cocktail several minutes before use; use on ice.

2. Membrane Protein Solubilization Buffer

Function: Membrane protein solubilization/extraction reagent; used to solubilize membrane proteins from the membrane pellet.

Stage of Use: After obtaining the membrane pellet.

Note: Thaw at room temperature and mix before use, then place on ice; add protease inhibitor cocktail freshly before use.

3. Protease Inhibitor Cocktail

Function: Inhibits protein degradation.

Stage of Use: Added freshly before use to Lysis Buffer and Membrane Protein Solubilization Buffer.

Note: Add according to product instructions; replaces PMSF with broader protection.

4. Phosphatase Inhibitor (Optional, user-supplied or included in kit)

Function: Protects phosphorylated proteins.

Stage of Use: Added to Lysis Buffer, Membrane Protein Solubilization Buffer, and homogenization buffer when extracting phosphorylated proteins.

Note: Use only when detecting phosphorylated proteins; add according to product instructions.

5. 5¡Á Loading Buffer

Function: Protein sample denaturation and loading.

Stage of Use: Downstream Western blot sample preparation.

Note: Add proportionally; conventional membrane proteins may be denatured by boiling; heat-sensitive membrane proteins are recommended to be incubated at 37¡ãC for 30 minutes.

Note: Lysis Buffer is not a membrane pellet resuspension buffer and should not be used to resuspend membrane pellets. Membrane Protein Solubilization Buffer is not a cell lysis buffer and should not be used for cell pretreatment. Follow the order specified in this manual.

Storage and Stability

1. Store all components at -20¡ãC, protected from light.

2. When stored as directed, the product is stable for 12 months from the date of manufacture.

3. Before use, thaw Lysis Buffer and Membrane Protein Solubilization Buffer at room temperature and mix; place on ice immediately after thawing.

4. Aliquot extracted protein fractions into single-use volumes and store at -70¡ãC to -80¡ãC. Avoid repeated freeze-thaw cycles.

5. Store 5¡Á Loading Buffer according to product instructions; check for precipitate before use. If present, dissolve by warming at 37¡ãC and mix.

Protocol

Important Notes

1. Perform all steps at 4¡ãC or on ice whenever possible.

2. Pre-chill PBS, centrifuge tubes, and glass homogenizers.

3. Add protease inhibitor cocktail freshly to Lysis Buffer and Membrane Protein Solubilization Buffer several minutes before use. If detecting phosphorylated proteins, also add phosphatase inhibitors.

4. For adherent cells, avoid trypsin digestion whenever possible to prevent trypsin-mediated degradation of membrane proteins; use a cell scraper or EDTA-containing, trypsin-free dissociation solution.

1. Reagent Preparation

1.1 Thaw and mix Lysis Buffer and Membrane Protein Solubilization Buffer at room temperature; place on ice immediately after thawing.

1.2 Prepare appropriate volumes of Lysis Buffer and Membrane Protein Solubilization Buffer.

1.3 Add protease inhibitor cocktail several minutes before use; if detecting phosphorylated proteins, also add phosphatase inhibitors.

1.4 Pre-chill PBS and homogenizer.

1.5 Prepare 5¡Á Loading Buffer as needed for downstream experiments.

2. Sample Preparation

2.1 Cultured Cells

Adherent cells:

(1) Culture approximately 20-50 million cells.

(2) Wash once with PBS; scrape cells with a cell scraper, or treat with EDTA-containing, trypsin-free dissociation solution until cells are no longer tightly adherent, and dislodge cells by pipetting.

(3) Collect cells by centrifugation, aspirate supernatant, and retain the cell pellet. Avoid trypsin digestion whenever possible.

(4) Gently resuspend the cell pellet in an appropriate volume of ice-cold PBS and count a small aliquot; centrifuge the remaining cells at 600 x g for 5 minutes at 4¡ãC to pellet.

(5) Discard supernatant; centrifuge at 600 x g for 1 minute at 4¡ãC to collect residual liquid from the tube wall and further pellet cells; aspirate residual liquid as thoroughly as possible.

Suspension cells:

(1) Culture approximately 20-50 million cells, collect directly by centrifugation, aspirate supernatant, and retain the cell pellet.

(2) Gently resuspend and wash with ice-cold PBS, centrifuge at 600 x g for 5 minutes at 4¡ãC, discard supernatant, and aspirate residual liquid thoroughly.

2.2 Tissue Samples

(1) Take approximately 100 mg of tissue and carefully mince into small pieces with scissors. For limited samples, 30-50 mg may be used with unchanged reagent volumes and protocol steps, though final membrane protein yield will be correspondingly reduced.

(2) Add 1 mL of Lysis Buffer supplemented with protease inhibitor cocktail (and phosphatase inhibitors if detecting phosphorylated proteins) immediately before use. Gently suspend tissue pieces and incubate on ice for 10-15 minutes.

Note: At this stage, Lysis Buffer serves as a cell lysis/pretreatment buffer to promote cell lysis and weaken cellular structures, while exhibiting low solubilizing capacity for membrane proteins and weak disruption of membrane structure to reduce premature release of membrane proteins.

3. Cell Pretreatment

(1) Add 1 mL of Lysis Buffer supplemented with protease inhibitor cocktail (and phosphatase inhibitors if detecting phosphorylated proteins) immediately before use to 20-50 million cells.

(2) Gently and thoroughly suspend cells and incubate on ice for 10-15 minutes.

(3) This step is Lysis Buffer pretreatment, which promotes cell lysis; avoid vigorous handling that may excessively disrupt membrane structure.

4. Cell or Tissue Disruption and Assessment of Disruption Efficiency

(1) Transfer the cell suspension or tissue sample to an appropriately sized ice-cold glass homogenizer and homogenize approximately 30-50 strokes.

(2) Homogenization efficiency depends on cell type and tissue type; the number of strokes required varies and should be optimized.

(3) Microscopic assessment: After 30 strokes, place approximately 2-3 µL of homogenate on a coverslip and observe under a microscope. If perinuclear halos or intact cell morphology are visible, cells remain intact. If 70-80% of cells lack perinuclear halos and intact morphology, cells are sufficiently disrupted and the next step may proceed; otherwise, homogenize an additional 10-30 strokes until at least 70% of cells are disrupted.

(4) Record the effective homogenization strokes and homogenizer used for this cell/tissue type for reference in subsequent experiments, avoiding repeated optimization.

(5) Without a glass homogenizer: For cultured cells, cells may be disrupted by freeze-thaw. Subject the sample from step 3 to two cycles of alternating liquid nitrogen and room temperature freeze-thaw; examine a small aliquot microscopically for disruption efficiency. If less than 70%, increase the number of freeze-thaw cycles until disruption exceeds 70%.

Note: After Lysis Buffer pretreatment, disruption efficiency should still be assessed microscopically. Different homogenizers and cell types require different numbers of strokes; record the conditions.

5. Removal of Nuclei and Unbroken Cells

(1) Centrifuge at 700 x g for 10 minutes at 4¡ãC.

(2) Carefully collect the supernatant into a new pre-chilled centrifuge tube. Do not contact the pellet when aspirating the supernatant.

(3) Leave approximately 30-50 µL of supernatant unaspirated to ensure higher purity of the collected supernatant.

6. Pellet Membrane Fragments

(1) Centrifuge at 14,000 x g for 30 minutes at 4¡ãC to pellet membrane fragments.

(2) The pellet is the membrane fragment fraction; the supernatant is the cytoplasmic protein fraction.

Important Note: The 14,000 x g centrifugation primarily pellets membrane fragments. If organelle membranes (e.g., mitochondrial, endoplasmic reticulum, Golgi, or lysosomal membranes) are to be collected, 14,000 x g is generally insufficient: the supernatant after high-speed centrifugation may still contain small organelles and membrane vesicles. In this case, before collecting cytoplasmic proteins, further ultracentrifuge the 14,000 x g supernatant (e.g., 100,000 x g or higher, 30-60 minutes, 4¡ãC) to obtain the organelle membrane fragment fraction. Specific centrifugation force and time should be optimized based on target organelles and vesicle size. If only membrane and cytoplasmic protein separation is required, 14,000 x g for 30 minutes is sufficient; if organelle membranes are also to be collected, it is recommended to explicitly add an ultracentrifugation step in the method and validate fraction purity with corresponding organelle markers.

7. Collection of Cytoplasmic Proteins

(1) The supernatant is the cytoplasmic protein fraction. If ultracentrifugation was performed as noted in step 6 to collect organelle membranes, the supernatant after ultracentrifugation is the cytoplasmic protein fraction.

(2) Leave 30-50 µL of supernatant unaspirated to avoid contacting the pellet and contaminating the supernatant sample.

(3) Aliquot cytoplasmic proteins and store at -70¡ãC to -80¡ãC. Avoid repeated freeze-thaw cycles.

(4) Expected yield: Approximately 5-30 mg of cytoplasmic protein can be obtained per 50 million cells using this product, varying by cell type.

8. Membrane Protein Extraction

(1) Centrifuge at 14,000 x g for 10 seconds at 4¡ãC and aspirate the supernatant as thoroughly as possible. The pellet may be lightly touched, and a very small amount of pellet may be aspirated.

(2) Add 200 µL of Membrane Protein Solubilization Buffer (may be increased to 300 µL if necessary). Add protease inhibitor cocktail freshly before use; if detecting phosphorylated proteins, also add phosphatase inhibitors.

(3) Resuspend the pellet by vigorous vortexing at maximum speed for 5 seconds, then incubate on ice for 5-10 minutes.

(4) Repeat the vortexing and ice incubation 1-2 times to fully extract membrane proteins.

(5) Centrifuge at 14,000 x g for 5 minutes at 4¡ãC and collect the supernatant as the membrane protein solution.

(6) Aliquot membrane protein fractions and store at -70¡ãC to -80¡ãC. Avoid repeated freeze-thaw cycles.

(7) Expected yield: Approximately 0.3-3 mg of membrane protein can be obtained per 50 million cells using this product, varying by cell type.

(8) For membrane proteins with special requirements, an appropriate user-prepared membrane protein solubilization buffer may be used for extraction.

Note: Membrane Protein Solubilization Buffer is a membrane protein solubilization/extraction reagent and should be used after obtaining the membrane pellet. Do not use Membrane Protein Solubilization Buffer for cell pretreatment or as a substitute for Lysis Buffer.

9. Use of 5¡Á Loading Buffer and Protein Denaturation

(1) According to downstream Western blot requirements, add 5¡Á Loading Buffer to the cytoplasmic or membrane protein fraction, typically at a 4:1 ratio (sample:5¡Á Loading Buffer), or adjust to a final 1¡Á concentration according to product instructions.

(2) Mix and denature according to standard conditions.

(3) Conventional membrane proteins: May be denatured by heating under standard laboratory conditions (e.g., boiling at 95-100¡ãC for 5-10 minutes); follow target protein and antibody instructions.

(4) Heat-sensitive membrane proteins: Boiling is not recommended. Use gentle denaturation conditions: 37¡ãC water bath for 30 minutes. After denaturation, centrifuge briefly and load the supernatant.

(5) If unsure whether the target membrane protein is heat-sensitive, first use gentle denaturation at 37¡ãC for 30 minutes; if signal is weak or aggregation occurs, optimize denaturation conditions within the range permitted by the antibody instructions.

(6) Denatured samples may be aliquoted and stored at -20¡ãC to -80¡ãC. Avoid repeated freeze-thaw cycles.

Note: 5¡Á Loading Buffer is used for downstream sample preparation and does not participate in membrane/cytoplasmic fraction separation. Gentle denaturation at 37¡ãC for 30 minutes is suitable for heat-sensitive membrane proteins; conventional membrane proteins may still be processed under standard conditions.

10. Quality Control and Downstream Analysis

(1) Quantify protein concentration in both cytoplasmic and membrane fractions (BCA or Bradford).

(2) Verify fraction purity by Western blot using compartment-specific markers:

Membrane fraction markers: Na⁺/K⁺-ATPase or cadherin.

Cytoplasmic fraction markers: GAPDH or ¦Â-tubulin.

If organelle membranes are collected, include corresponding markers: e.g., COX IV for mitochondria, Calnexin for endoplasmic reticulum, GM130 for Golgi.

(3) Ideal results: Membrane fraction is enriched for corresponding markers, cytoplasmic fraction is enriched for GAPDH/¦Â-tubulin, with low cross-contamination.

(4) If membrane markers are evident in the cytoplasmic fraction, check whether 14,000 x g centrifugation was sufficient and whether the pellet was touched when aspirating supernatant; if necessary, increase membrane pelleting centrifugation force or add a membrane pellet washing step.

(5) If cytoplasmic markers are evident in the membrane fraction, gently wash the membrane pellet once with ice-cold PBS or homogenization buffer, then aspirate thoroughly before membrane protein solubilization.

(6) When detecting phosphorylated proteins, confirm that both protease inhibitor cocktail and phosphatase inhibitors have been added, minimize operation time, and maintain low temperature.

(7) When using 5¡Á Loading Buffer, note that heat-sensitive membrane proteins should be incubated at 37¡ãC for 30 minutes to avoid aggregation or degradation caused by high temperature.

Precautions

1. Perform all steps at 4¡ãC or on ice whenever possible to prevent protein degradation.

2. Add protease inhibitor cocktail freshly before use; if detecting phosphorylated proteins, also add phosphatase inhibitors.

3. For adherent cells, avoid trypsin digestion whenever possible to prevent trypsin-mediated degradation of membrane proteins.

4. Lysis Buffer is a cell lysis/pretreatment buffer with low solubilizing capacity for membrane proteins and weak disruption of membrane structure; Membrane Protein Solubilization Buffer is a membrane protein solubilization/extraction reagent. The two have different functions and their stages of use cannot be interchanged.

5. Homogenization conditions should be optimized for cell type and tissue type, using microscopic criteria of 70-80% cell disruption, and the number of strokes and homogenizer should be recorded.

6. Do not contact the pellet when aspirating supernatant; leaving 30-50 µL of residual liquid ensures purity.

7. Membrane pellet centrifugation conditions are 14,000 x g for 30 minutes; if organelle membranes are to be collected, further ultracentrifugation (e.g., 100,000 x g, 30-60 minutes, 4¡ãC) is required after high-speed centrifugation, with validation using corresponding markers.

8. Aliquot extracted fractions into single-use volumes and store at -70¡ãC to -80¡ãC. Avoid repeated freeze-thaw cycles.

9. When using 5¡Á Loading Buffer, conventional membrane proteins may be denatured under standard conditions; heat-sensitive membrane proteins are recommended to be incubated at 37¡ãC for 30 minutes to avoid aggregation or degradation caused by boiling.

10. This product is for research use only and is not intended for diagnostic or therapeutic procedures.

FAQ

Q1: What is the difference between Lysis Buffer and Membrane Protein Solubilization Buffer?

A1: Lysis Buffer is a cell lysis/pretreatment buffer used before homogenization to promote cell lysis, with low solubilizing capacity for membrane proteins and weak disruption of membrane structure. Membrane Protein Solubilization Buffer is a membrane protein solubilization/extraction reagent used after obtaining the membrane pellet. The two have different stages of use and functions and cannot be interchanged.

Q2: How can I determine whether cells are sufficiently disrupted?

A2: Examine 2-3 µL of homogenate microscopically. If 70-80% of cells lack perinuclear halos and intact morphology, disruption is sufficient; otherwise, continue homogenizing for 10-30 strokes. Without a glass homogenizer, cultured cells may be subjected to repeated liquid nitrogen/room temperature freeze-thaw cycles until disruption exceeds 70% by microscopic assessment.

Q3: Why should 30-50 µL of supernatant be left unaspirated?

A3: To avoid contacting the pellet, reduce contamination from membrane fragments or nuclei, and ensure cytoplasmic fraction purity.

Q4: What should I do if membrane protein yield is low?

A4: Check whether homogenization was sufficient, membrane pelleting centrifugation was complete, and whether vortexing was vigorous and repeated 1-2 times during membrane protein solubilization. If necessary, increase Membrane Protein Solubilization Buffer volume to 300 µL, or optimize the solubilization buffer for special membrane proteins. If loss of small membrane vesicles is suspected, increase membrane pelleting centrifugation force or add an ultracentrifugation step, with marker validation.

Q5: How can I verify membrane/cytoplasmic fraction purity?

A5: Western blot is recommended: use Na⁺/K⁺-ATPase or cadherin for the membrane fraction, and GAPDH or ¦Â-tubulin for the cytoplasmic fraction. If organelle membranes are collected, include corresponding organelle markers. If cross-contamination is evident, check supernatant aspiration technique and membrane pelleting centrifugation conditions.

Q6: What should I note when detecting phosphorylated proteins?

A6: In addition to protease inhibitor cocktail, add phosphatase inhibitors to Lysis Buffer, Membrane Protein Solubilization Buffer, and homogenization buffer; maintain low temperature throughout, minimize operation time, and aliquot for storage to avoid repeated freeze-thaw cycles.

Q7: How should I collect organelle membrane structures?

A7: 14,000 x g for 30 minutes primarily pellets membrane fragments. To collect organelle membranes, further ultracentrifuge the 14,000 x g supernatant (e.g., 100,000 x g, 30-60 minutes, 4¡ãC); the pellet is enriched in organelle membranes, and the supernatant is cytoplasmic protein. Specific conditions should be optimized for target organelles and validated with corresponding markers.

Q8: How should 5¡Á Loading Buffer be used, and how should heat-sensitive membrane proteins be denatured?

A8: Add 5¡Á Loading Buffer to the protein sample to a final 1¡Á concentration and mix. Conventional membrane proteins may be denatured by standard heating; heat-sensitive membrane proteins are recommended to be incubated at 37¡ãC for 30 minutes to avoid aggregation or degradation caused by boiling. After denaturation, centrifuge briefly and load the supernatant. If unsure whether the target protein is heat-sensitive, gentle denaturation at 37¡ãC for 30 minutes is preferred.

Disclaimer

1. This product is for research use only and is not intended for diagnostic or therapeutic procedures.

2. Due to the variable nature of biological research, optimization of extraction conditions is recommended for specific cell types and tissues.

3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages.

4. Wear appropriate protective clothing and gloves when handling this product.

Ordering Information

Catalog Number: IC-7910

Product Name: Cell Membrane and Cytoplasmic Protein Extraction Kit

Size: 25 extractions / 50 extractions / 100 extractions

Suggested Retail Price:

25 extractions: CNY ¥1500.00 / USD $150.00 / EUR €180.00 / JPY ¥27000.00

50 extractions: CNY ¥2250.00 / USD $225.00 / EUR €270.00 / JPY ¥40500.00

100 extractions: CNY ¥3550.00 / USD $355.00 / EUR €426.00 / JPY ¥63900.00

Note: The specific price is subject to the transaction price in the offline contract. This document is provided for technical reference only and does not constitute any form of sales advertisement or invitation to offer. For product details and purchase inquiries, please contact local sales representatives.