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Cell Fractionation and Organelle Isolation |
Cell fractionation and organelle isolation are essential techniques in biological research involved the separation of cellular components for downstream assays including western blotting, protein assays, and enzyme activity assays. Cell fractionation is the breakdown of cells into distinct components such as cytoplasmic, membrane, nuclear, chromatin-bound, and cytoskeletal proteins. On the other hand, organelle isolation focuses on the separation of subcellular structures like mitochondria, lysozyme, and synaptosomes. These methods allow for the comprehensive analysis of cellular structures along with enabling a deeper understanding of cellular functions and interactions within the complex microenvironment of the cell.
Thermo Fisher Scientific offers cell fractionation and organelle isolation kits and reagents that are optimized for stepwise separation, enrichment, and extraction of proteins. These products are specifically designed for quick and efficient fractionation or isolation of cells and tissues, resulting in high protein yields within 1-3 hours. Choose from our comprehensive range of cell fractionation or organelle isolation product based on your specific separation needs.
Learn more: Cell Fractionation and Organelle Isolation
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NE-PER Nuclear and Cytoplasmic Extraction Reagents | Subcellular Protein Fractionation Kit for cultured cells | Subcellular Protein Fractionation Kit for tissues | |
When to use (compatible cell structure) | Nucleus, cytoplasm | Nucleus, cytoplasm, membrane, cytoskeletal, chromatin-bound | |
Compatible sample types | Tissues and cultured mammalian cells | Tissues or cultured mammalian cells | |
Sample processing time | 2 hours | 2–3 hours | |
Mechanical disruption required? | Yes, for tissue | Yes, for tissue | |
Amount of sample processed | 50 samples with 2 million cells (20 µL packed) | 25 extractions of 200 mg tissue or 50 samples with 2 million cells (20 µL packed) | |
Compatible protein assays | BCA Protein Assay (CER dilute 1:4), Bradford Assays (CER dilute 1:4) | BCA Protein Assay (all fractions), Bradford Assays (NEB fraction) | |
Downstream compatibility | Western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling CER fraction only: RNA EMSA, kinase assays, RT-PCR | IP, western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling | |
Protease or phosphatase inhibitors recommended? | Yes | Protease inhibitors included in kit | |
Available size(s) | 15 mL 75 mL | 1 kit (50 samples) | 1 kit (25 samples) |
User guide | User Guide: NE-PER Nuclear and Cytoplasmic Extraction Reagents | User Guide: Subcellular Protein Fractionation Kit for Cultured Cell | User Guide: Subcellular Protein Fractionation Kit for Tissue |
A variety of methods exist to isolate nuclei and prepare nuclear protein extracts. However, most of these are lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation, or dialysis steps that may compromise the integrity of many fragile nuclear proteins. The NE-PER Nuclear and Cytoplasmic Extraction Kit generates both functional cytoplasmic and nuclear protein fractions recovered from the same cell (Figure 1) or tissue (Figure 2) sample in less than two hours. The simple protocol involves a stepwise lysis that separates the cytoplasm from intact nuclei, and then extracts nuclear protein away from genomic DNA and mRNA. Yield from two million cells typically includes 200 to 500 µg of cytoplasmic protein and 100 to 200 µg of nuclear protein (at a concentration of 1 mg/mL). Additionally, cross-contamination between cytoplasmic and nuclear fractions has been shown to be less than 10% (data not shown).
Figure 1. Total protein profile of cytoplasmic and nuclear extracts prepared from different mammalian cell lines. Mammalian cells were harvested, rinsed with PBS, and lysed using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit. Extracts were quantified using the Pierce 660nm Protein Assay Reagent. Values are the average of two separate isolations.
Separation of proteins by subcellular isolation is one of the methods to enrich for proteins while maintaining the ability to characterize biological proteins and study protein translocation. Differential detergent extraction is a classic biochemical technique used to separate proteins based on sequential solubilization of cellular compartments using different detergents. The Thermo Scientific Subcellular Protein Fractionation Kit provides a combination of reagents for stepwise lysis of cells into cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal protein fractions that are functional (Figure3, 4). Cross-contamination between extracts from each subcellular fractions is generally less than 15% (data not shown).
Learn more: Subcellular protein fractionation to enhance proteomic coverage of cultured cells
Figure 3. Schematic overview of the subcellular fractionation procedure. Cellular compartments are sequentially extracted by incubating cells with cytoplasmic extraction buffer (CEB), followed by membrane extraction buffer (MEB) and nuclear extraction buffer (NEB). Adding micrococcal nuclease (MNase) to NEB extracts chromatin-bound proteins from the cell pellet before adding the pellet extraction buffer (PEB) to solubilize cytoskeletal proteins.
Figure 4. Western blots of fractionated cellular proteins. HeLa cells (2 x 10^6) were fractionated using the Subcellular Protein Fractionation Kit. Normalized portions of each extract (10 µg) were analyzed by Western blotting using specific antibodies against proteins from various cellular compartments including cytoplasmic (HSP90), plasma membrane (EGFR), endoplasmic reticulum (calreticulin), nuclear soluble (SP1 and HDAC2); chromatin-bound (histone 3); and cytoskeleton (cytokeratin 18 and vimentin). The blots were probed with goat anti-rabbit (H+L) HRP or goat anti-mouse (H+L) HRP and detected with Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Part No. 34076). The results demonstrate clear fractionation of each protein with low cross contamination among fractions.
CE: cytoplasmic extract
ME: membrane extract
NE: nuclear extract
CB: chromatin-bound extract
PE: pellet extract
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Mitochondria isolation kit for tissue | Mitochondria isolation kit for cultured cells | Lysosome enrichment kit for tissues and cultured cells | |
When to use (compatible cell organelle) | Mitochondria | Lysosome | |
Compatible sample types | Heart and liver tissue | Cultured mammalian cells | Tissue and cultured cells |
Sample processing time | 1 hour | 40 mins | 2 hours |
Mechanical disruption required? | Dounce optional | Dounce optional | Yes: Dounce, sonication, or polytron tissue tearer |
Amount of sample processed | 50 samples each containing 50–200 mg of soft or hard tissue | 50 samples each containing 20 million cultured mammalian cells | 25 samples each containing 50–200 mg of cells or tissue |
Compatible protein assays | BCA Protein Assays, Bradford Assays | BCA Protein Assays, Detergent Compatible Bradford | Bradford Assays |
Downstream compatibility | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies |
Protease or phosphatase inhibitors recommended? | Yes, both | Yes, both | Yes, EDTA free |
Available size(s) | 1 kit (50 isolations) | 1 kit (50 isolations) | 1 kit (25 samples of 50 to 200 mg cells or tissues each) |
User guide | User Guide: Mitochondria Isolation Kit for Tissue | User Guide: Mitochondria Isolation Kit for Cultured Cells | User Guide: Lysosome Enrichment Kit for Tissue and Cultured Cells |
The mitochondria isolation kits for tissue or cultured cells include optimized reagents and protocols that enable high yields of pure mitochondria. Additionally, they each use differential centrifugation to separate intact mitochondria.
The Mitochondria Isolation Kit for Tissue offers two streamlined methods, a reagent-based or Dounce homogenization procedure, to isolate mitochondria from soft and hard tissue samples in about an hour (Figure 5). The reagent-based option enables the simultaneous processing of multiple samples.
The Mitochondria Isolation Kit for Cultured Cells primarily uses a reagent-based method to isolate mitochondria in approximately 40 minutes (Figure 6). Like the mitochondria isolation kit for tissues, this reagent-based method can be used to process multiple samples at the same time. If greater yield is needed, the mitochondria isolation kit for cultured cells also offers an optimized Dounce homogenization procedure that results in 2-fold greater mitochondria recovery.
Figure 5. Integrity of mitochondria extracted from tissues. Mitochondria (M) and cytosolic (C) fractions were prepared from fresh rat liver (Panels 1, 2, 3, 5 and 6) and heart (Panel 4) tissue samples using the reagent-based and Dounce homogenization methods of the Thermo Scientific Mitochondria Isolation Kit for Tissue. Fractions were analyzed via Western blot for COX4, VDAC and Cytochrome C mitochondrial proteins. Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate was used for detection. COX4 is an inner-mitochondria membrane protein, VDAC is an outer-mitochondria membrane protein and cytochrome C is located in the intermembrane space. Results indicate that cytosolic proteins were thoroughly separated from the isolated mitochondria.
Figure 6. Integrity of mitochondria extracted from cultured cells. C6 cells were extracted with Thermo Scientific Mitochondria Isolation Kit using the reagent-based method (A and B) or Dounce homogenization (C and D). Mitochondrial (M) and cytosolic (C) fractions were analyzed via western blot for cytochrome C (A and C) or voltage-dependent anion channel (VDAC) (B and D). Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate was used for detection. Results indicate that mitochondria remained intact.
The Lysosome Enrichment Kit for Tissues and Cultured Cells allows for the isolation and enrichment of intact lysosomes from cells (Figure 7) and soft and hard tissue samples. Similar to the mitochondria isolation kits, the lysosome enrichment kit uses reagent-based or Dounce homogenization method. For the reagent-based procedure, the kit contains OptiPrep Cell Separation Media to isolate and enrich lysosomes from the contaminating cell structures.
Figure 7. Lysosome enrichment from cultured cells. Approximately 200 mg of wet cell paste was processed from A431 and HeLa cells using the Lysosome Enrichment Kit for Tissue and Cultured Cells. Total cell lysate and isolated lysosomes were analyzed by Western blotting for Lamp-1 and Cathepsin D, membrane-bound and soluble lysosome markers, respectively.
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Syn-PER Synaptic Protein Extraction Reagent | ||
When to use (compatible cell organelle) | Synaptic proteins | Synaptosomes |
Compatible sample types | Brain tissue and primary cultured neurons | |
Sample processing time | <1 hour | |
Mechanical disruption required? | Yes, dounce for tissue | |
Amount of sample processed | 10 g neuronal tissue or primary cultured neurons per 100 mL | |
Compatible protein assays | BCA Protein Assays | |
Downstream compatibility | western blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitations | neurotransmitter release assay only |
Recommended supplemental protease/phosphatase inhibitors | Yes, EDTA free | |
Available size(s) | 100 mL | |
User guide | User Guide: Syn-PER Synaptic Protein Extraction Reagent |
Synaptosomes are isolated nerve terminals that are generated during the homogenization of nerve tissue. Syn-PER Synaptic Protein Extraction Reagent is specifically formulated to gently and rapidly extract synaptosomes from neuronal tissue (Figure 8) and primary cultured neurons with high yield. Synaptosomes are commonly used to study synaptic function such as neurotransmitter release, identification of changes in protein composition and function in synapses, and protein phosphorylation events associated with synapses.
The Syn-PER reagent can be used to isolate functional pre- and post-synaptic proteins (i.e., intact membranes and protein complexes of synapses) and allows for the preservation of phosphoprotein integrity. Although, this reagent does not contain protease or phosphatase inhibitors, Halt Protease and/or Phosphatase Inhibitors can be added before use to prevent proteolysis or for additional protection from the high phosphatase activity present in brain tissue.
Methods article: Method to isolate functional synaptosomes
Figure 8. Synaptic protein enrichment in brain tissue. Greater enrichment of synaptic proteins is achieved in samples prepared using the Thermo Scientific Syn-PER Reagent than with a homebrew reagent. Total protein (10 µg) from mouse brain tissue homogenates (H), cytosol (C) fraction, and synaptosome (Syn) suspension were analyzed by western blot. The total protein yield in the synaptosome suspension produced from fresh mouse brain (200 mg) was three-fold greater using the Syn-PER Reagent compared to a typical homebrew reagent.
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NE-PER Nuclear and Cytoplasmic Extraction Reagents | Subcellular Protein Fractionation Kit for cultured cells | Subcellular Protein Fractionation Kit for tissues | |
When to use (compatible cell structure) | Nucleus, cytoplasm | Nucleus, cytoplasm, membrane, cytoskeletal, chromatin-bound | |
Compatible sample types | Tissues and cultured mammalian cells | Tissues or cultured mammalian cells | |
Sample processing time | 2 hours | 2–3 hours | |
Mechanical disruption required? | Yes, for tissue | Yes, for tissue | |
Amount of sample processed | 50 samples with 2 million cells (20 µL packed) | 25 extractions of 200 mg tissue or 50 samples with 2 million cells (20 µL packed) | |
Compatible protein assays | BCA Protein Assay (CER dilute 1:4), Bradford Assays (CER dilute 1:4) | BCA Protein Assay (all fractions), Bradford Assays (NEB fraction) | |
Downstream compatibility | Western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling CER fraction only: RNA EMSA, kinase assays, RT-PCR | IP, western blot, ELISA, EMSA, reporter assays, enzyme assays, amine reactive labeling | |
Protease or phosphatase inhibitors recommended? | Yes | Protease inhibitors included in kit | |
Available size(s) | 15 mL 75 mL | 1 kit (50 samples) | 1 kit (25 samples) |
User guide | User Guide: NE-PER Nuclear and Cytoplasmic Extraction Reagents | User Guide: Subcellular Protein Fractionation Kit for Cultured Cell | User Guide: Subcellular Protein Fractionation Kit for Tissue |
A variety of methods exist to isolate nuclei and prepare nuclear protein extracts. However, most of these are lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation, or dialysis steps that may compromise the integrity of many fragile nuclear proteins. The NE-PER Nuclear and Cytoplasmic Extraction Kit generates both functional cytoplasmic and nuclear protein fractions recovered from the same cell (Figure 1) or tissue (Figure 2) sample in less than two hours. The simple protocol involves a stepwise lysis that separates the cytoplasm from intact nuclei, and then extracts nuclear protein away from genomic DNA and mRNA. Yield from two million cells typically includes 200 to 500 µg of cytoplasmic protein and 100 to 200 µg of nuclear protein (at a concentration of 1 mg/mL). Additionally, cross-contamination between cytoplasmic and nuclear fractions has been shown to be less than 10% (data not shown).
Figure 1. Total protein profile of cytoplasmic and nuclear extracts prepared from different mammalian cell lines. Mammalian cells were harvested, rinsed with PBS, and lysed using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit. Extracts were quantified using the Pierce 660nm Protein Assay Reagent. Values are the average of two separate isolations.
Separation of proteins by subcellular isolation is one of the methods to enrich for proteins while maintaining the ability to characterize biological proteins and study protein translocation. Differential detergent extraction is a classic biochemical technique used to separate proteins based on sequential solubilization of cellular compartments using different detergents. The Thermo Scientific Subcellular Protein Fractionation Kit provides a combination of reagents for stepwise lysis of cells into cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal protein fractions that are functional (Figure3, 4). Cross-contamination between extracts from each subcellular fractions is generally less than 15% (data not shown).
Learn more: Subcellular protein fractionation to enhance proteomic coverage of cultured cells
Figure 3. Schematic overview of the subcellular fractionation procedure. Cellular compartments are sequentially extracted by incubating cells with cytoplasmic extraction buffer (CEB), followed by membrane extraction buffer (MEB) and nuclear extraction buffer (NEB). Adding micrococcal nuclease (MNase) to NEB extracts chromatin-bound proteins from the cell pellet before adding the pellet extraction buffer (PEB) to solubilize cytoskeletal proteins.
Figure 4. Western blots of fractionated cellular proteins. HeLa cells (2 x 10^6) were fractionated using the Subcellular Protein Fractionation Kit. Normalized portions of each extract (10 µg) were analyzed by Western blotting using specific antibodies against proteins from various cellular compartments including cytoplasmic (HSP90), plasma membrane (EGFR), endoplasmic reticulum (calreticulin), nuclear soluble (SP1 and HDAC2); chromatin-bound (histone 3); and cytoskeleton (cytokeratin 18 and vimentin). The blots were probed with goat anti-rabbit (H+L) HRP or goat anti-mouse (H+L) HRP and detected with Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Part No. 34076). The results demonstrate clear fractionation of each protein with low cross contamination among fractions.
CE: cytoplasmic extract
ME: membrane extract
NE: nuclear extract
CB: chromatin-bound extract
PE: pellet extract
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Mitochondria isolation kit for tissue | Mitochondria isolation kit for cultured cells | Lysosome enrichment kit for tissues and cultured cells | |
When to use (compatible cell organelle) | Mitochondria | Lysosome | |
Compatible sample types | Heart and liver tissue | Cultured mammalian cells | Tissue and cultured cells |
Sample processing time | 1 hour | 40 mins | 2 hours |
Mechanical disruption required? | Dounce optional | Dounce optional | Yes: Dounce, sonication, or polytron tissue tearer |
Amount of sample processed | 50 samples each containing 50–200 mg of soft or hard tissue | 50 samples each containing 20 million cultured mammalian cells | 25 samples each containing 50–200 mg of cells or tissue |
Compatible protein assays | BCA Protein Assays, Bradford Assays | BCA Protein Assays, Detergent Compatible Bradford | Bradford Assays |
Downstream compatibility | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, ELISA, amine reactive labeling, apoptosis, signal transduction, and metabolic studies | Western blot, 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies |
Protease or phosphatase inhibitors recommended? | Yes, both | Yes, both | Yes, EDTA free |
Available size(s) | 1 kit (50 isolations) | 1 kit (50 isolations) | 1 kit (25 samples of 50 to 200 mg cells or tissues each) |
User guide | User Guide: Mitochondria Isolation Kit for Tissue | User Guide: Mitochondria Isolation Kit for Cultured Cells | User Guide: Lysosome Enrichment Kit for Tissue and Cultured Cells |
The mitochondria isolation kits for tissue or cultured cells include optimized reagents and protocols that enable high yields of pure mitochondria. Additionally, they each use differential centrifugation to separate intact mitochondria.
The Mitochondria Isolation Kit for Tissue offers two streamlined methods, a reagent-based or Dounce homogenization procedure, to isolate mitochondria from soft and hard tissue samples in about an hour (Figure 5). The reagent-based option enables the simultaneous processing of multiple samples.
The Mitochondria Isolation Kit for Cultured Cells primarily uses a reagent-based method to isolate mitochondria in approximately 40 minutes (Figure 6). Like the mitochondria isolation kit for tissues, this reagent-based method can be used to process multiple samples at the same time. If greater yield is needed, the mitochondria isolation kit for cultured cells also offers an optimized Dounce homogenization procedure that results in 2-fold greater mitochondria recovery.
Figure 5. Integrity of mitochondria extracted from tissues. Mitochondria (M) and cytosolic (C) fractions were prepared from fresh rat liver (Panels 1, 2, 3, 5 and 6) and heart (Panel 4) tissue samples using the reagent-based and Dounce homogenization methods of the Thermo Scientific Mitochondria Isolation Kit for Tissue. Fractions were analyzed via Western blot for COX4, VDAC and Cytochrome C mitochondrial proteins. Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate was used for detection. COX4 is an inner-mitochondria membrane protein, VDAC is an outer-mitochondria membrane protein and cytochrome C is located in the intermembrane space. Results indicate that cytosolic proteins were thoroughly separated from the isolated mitochondria.
Figure 6. Integrity of mitochondria extracted from cultured cells. C6 cells were extracted with Thermo Scientific Mitochondria Isolation Kit using the reagent-based method (A and B) or Dounce homogenization (C and D). Mitochondrial (M) and cytosolic (C) fractions were analyzed via western blot for cytochrome C (A and C) or voltage-dependent anion channel (VDAC) (B and D). Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate was used for detection. Results indicate that mitochondria remained intact.
The Lysosome Enrichment Kit for Tissues and Cultured Cells allows for the isolation and enrichment of intact lysosomes from cells (Figure 7) and soft and hard tissue samples. Similar to the mitochondria isolation kits, the lysosome enrichment kit uses reagent-based or Dounce homogenization method. For the reagent-based procedure, the kit contains OptiPrep Cell Separation Media to isolate and enrich lysosomes from the contaminating cell structures.
Figure 7. Lysosome enrichment from cultured cells. Approximately 200 mg of wet cell paste was processed from A431 and HeLa cells using the Lysosome Enrichment Kit for Tissue and Cultured Cells. Total cell lysate and isolated lysosomes were analyzed by Western blotting for Lamp-1 and Cathepsin D, membrane-bound and soluble lysosome markers, respectively.
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Syn-PER Synaptic Protein Extraction Reagent | ||
When to use (compatible cell organelle) | Synaptic proteins | Synaptosomes |
Compatible sample types | Brain tissue and primary cultured neurons | |
Sample processing time | <1 hour | |
Mechanical disruption required? | Yes, dounce for tissue | |
Amount of sample processed | 10 g neuronal tissue or primary cultured neurons per 100 mL | |
Compatible protein assays | BCA Protein Assays | |
Downstream compatibility | western blot, enzymatic activity assays, protein-protein interaction studies, immunoprecipitations | neurotransmitter release assay only |
Recommended supplemental protease/phosphatase inhibitors | Yes, EDTA free | |
Available size(s) | 100 mL | |
User guide | User Guide: Syn-PER Synaptic Protein Extraction Reagent |
Synaptosomes are isolated nerve terminals that are generated during the homogenization of nerve tissue. Syn-PER Synaptic Protein Extraction Reagent is specifically formulated to gently and rapidly extract synaptosomes from neuronal tissue (Figure 8) and primary cultured neurons with high yield. Synaptosomes are commonly used to study synaptic function such as neurotransmitter release, identification of changes in protein composition and function in synapses, and protein phosphorylation events associated with synapses.
The Syn-PER reagent can be used to isolate functional pre- and post-synaptic proteins (i.e., intact membranes and protein complexes of synapses) and allows for the preservation of phosphoprotein integrity. Although, this reagent does not contain protease or phosphatase inhibitors, Halt Protease and/or Phosphatase Inhibitors can be added before use to prevent proteolysis or for additional protection from the high phosphatase activity present in brain tissue.
Methods article: Method to isolate functional synaptosomes
Figure 8. Synaptic protein enrichment in brain tissue. Greater enrichment of synaptic proteins is achieved in samples prepared using the Thermo Scientific Syn-PER Reagent than with a homebrew reagent. Total protein (10 µg) from mouse brain tissue homogenates (H), cytosol (C) fraction, and synaptosome (Syn) suspension were analyzed by western blot. The total protein yield in the synaptosome suspension produced from fresh mouse brain (200 mg) was three-fold greater using the Syn-PER Reagent compared to a typical homebrew reagent.
See also: Protein purification reagents and kits
The Protein Preparation Handbook provides useful information on our broad selection of reagents and tools for protein extraction, clean-up, immunoprecipitation, and purification. Practical information, selection guides, and relevant data are included to help you improve your protein yield and downstream analysis.
This modular, animated, and narrated eLearning course was developed to provide a succinct, contextual summary of common laboratory methods and techniques required to achieve optimal results in protein assays and experiments. There are knowledge checks throughout the course to test what you have learned.
For Research Use Only. Not for use in diagnostic procedures.