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Minute™ Antibody Enhancer

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Minute™ Antibody Enhancer (30 ml)

Cat #: WA-002

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Description

Minute™ Antibody Enhancer is able to significantly increase the sensitivity of commonly used immunoassays such as Western blotting, ELISA and immunohistochemistry. The enhancer can reduce the amount of primary and secondary antibodies used due to higher antibody dilution employed. The signal intensity can be increased in immunostaining applications with chromogenic or fluorescent substrates.

Kit includes:

Item

Quantity

Antibody Enhancer

30 ml
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Minute™ Anti-Clumping Nuclei Storage Buffer

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Minute™ Anti-Clumping Nuclei Storage Buffer (10 ml)

Cat #: WA-014

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Description

The traditional method for nuclei isolation from animal cells/tissues depends on the use of non-ionic detergent to lyse the plasma membrane and release nuclei which can be subsequently isolated by low-speed centrifugation and further purified by density gradient. One major problem associated with the traditional method is the clumping of nuclei after isolation, which, in some applications such as DNA and RNA extraction, may not be a problem. However, in other applications such as single nucleus sequencing or proteomics, clumping of isolated nuclei represents a major problem. The anti-clumping nuclei storage buffer is designed to address this issue. Nuclei resuspended in this buffer show significantly reduced clumping as compared to those resuspended in PBS with 5% BSA. The nucleus suspension can be stored at 4oC for days without significant aggregation and change in morphology. The nuclei can also be frozen in the buffer at -80oC or in dry ice for transportation or long-term storage.

For spin column-based single nuclei isolation, click here for the list of available products.

Comparison of the Storage Buffer with 5% BSA in PBS for Anti-Clumping Efficacy. Nuclei of frozen mouse spleen and lung tissues were isolated by Nucleus Isolation Kit for Tissues/cells (Cat# SN-047, Invent Biotechnologies, Plymouth MN). The isolated nuclei pellets were resuspended in 0.4 ml nuclei storage buffer or 0.4 ml 5% BSA in PBS and incubated at 4oC overnight.  The nuclei were resuspended by vortexing briefly and stained with trypan blue to compare the nuclear morphologies.

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Minute™ Detergent-Free Single Nuclei Isolation Kit

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Minute™ Detergent-Free Single Nuclei Isolation Kit (20 Preps)

Cat #: NI-024

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Description

Single-cell genomics requires high-quality and complete genetic materials from individual cells. However, intact single cells are hard to isolate from tissues, especially when frozen. In addition, dissociation-induced (either mechanical or enzymatic) population and expression bias are also problematic. It is more feasible to isolate intact nuclei to be used as the starting material for most single-cell genomics applications. 

The detergent-free nuclei isolation kit is designed to rapidly isolate intact nuclei from animal cultured cells or tissues (fresh or frozen). Intact nuclei can be isolated from the samples using proprietary spin-column-based technologies in less than 20 min without using tissue homogenizer and any detergents. The traditional method for nuclei isolation involves the use of non-ionic detergent, which has a tendency to cause unwanted nuclear aggregation. It is unclear why some nuclei are aggregated while others are not. It is also unknown whether non-aggregated nuclei are an unbiased representation of the whole nuclei population. Detergents also have the potential to damage the nuclear envelope resulting in leakage of nuclear matrix materials. For some cell/tissue types, nuclear envelop proteins could also be stripped off by the detergents. MinuteTM Detergent-Free Nuclei Isolation Kit provides a whole new way for nuclei isolation in contrast to traditional methods.

How it works: cells/tissues are first sensitized by buffer A before passing through the proprietary filter in a zigzag manner when high-speed centrifugal force is applied. The cells are ruptured when passing through the filter leaving intact native nuclei in the flow-through. The nuclei are separated from other small cell debris by low-speed centrifugation using the proprietary buffer B. The native and intact nuclei isolated can be used for a variety of downstream applications that include but not limited to: FACS analysis, single nucleus analysis (such as RNA-seq and ATAC-seq), immunofluorescence staining, cell cycle analysis, and/or apoptosis research.

For storage and transportation of isolated single nuclei, the following buffer is recommended:

WA-014: Minute™ Anti-Clumping Nuclei Storage Buffer 

NOTE: We also offer the following single nucleus isolation kits with a much cleaner background if the presence of detergent is not a concern:

SN-047: Minute™ Single Nucleus Isolation Kit for Tissues/Cells

BN-020: Minute™ Single Nucleus Isolation Kit for Neuronal Tissues/Cells

AN-029: Minute™ Nuclei and Cytosol Isolation Kit for Adipose Tissues/Cultured Adipocytes

Kit includes:

Items

Quantity

Buffer A

15 ml

Buffer B

30 ml

Filter Cartridges

20 units

Collection Tubes

20 units

Pestles

2 units

 

Nuclei Isolated from zebrafish brain tissue Using NI-024 and stained with DAPI (False-coloured in green) (Courtesy Sema Elif Eski, Singh Lab, Université Libre de Bruxelles).

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Minute™ Single Cell Isolation Kit for Fresh and Fixed Tissues

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Minute™ Single Cell Isolation Kit for Fresh and Fixed Tissues (50 Preps)

Cat #: SC-012

Description

MinuteTM single cell isolation kit comprises optimized tissue disaggregation buffers and specially designed filter cartridges with 2.0 ml collection tubes. The kit is designed to isolate single cells from fresh animal tissues rapidly, and it can also isolate nuclei from tissues briefly fixed with formaldehyde for chromosome immunoprecipitation (ChIP). The tissue disaggregation buffers are formulated to disaggregate animal tissues gently. The buffers don’t contain any proteinases and EDTA that may adversely affect cell surface markers. Due to filter cartridges with pre-defined pore size and a specially formulated buffer system, single-cell suspension can be obtained from fresh tissues in less than 10 min. This kit is specifically designed for lymphoid tissues such as the spleen, thymus, and lymph node. For other tissues, the performance is tissue type-dependent. For cell suspension isolation from frozen/fresh tissues, please refer to Cat# CS-031.

Kit includes:

Items

Quantity

Buffer A (for non-fixed tissues)

25 ml

Buffer B (for fixed tissues)

25 ml

Cell Isolation Filter Cartridges

50 units

Collection Tubes with Caps

50 units

Plastic Rods

2 units

 

References (8)
  1. Kuo C-T, et al. (2016). Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots. Nature Communications. http://www.nature.com/naturecommunications.
  2. Bultmann-Mellin, I., Dinger, K., Debuschewitz, C., Loewe, K. M., Melcher, Y., Plum, M. T., … & Jüngst, C. (2017). Role of LTBP-4 in alveolarization, angiogenesis and fibrosis in lungs. American Journal of Physiology-Lung Cellular and Molecular Physiology, ajplung-00031.
  3. Vergara, M. N., Flores-Bellver, M., Aparicio-Domingo, S., McNally, M., Wahlin, K. J., Saxena, M. T., … & Canto-Soler, M. V. (2017). Enabling quantitative screening in retinal organoids: 3D automated reporter quantification technology (3D-ARQ). Development, dev-146290.
  4. Janicova, A., Becker, N., Xu, B., Wutzler, S., Vollrath, T., Hildebrand, F., … & Relja, B. (2019). Endogenous uteroglobin as intrinsic anti-inflammatory signal modulates monocyte and macrophage subsets distribution upon sepsis induced lung injury. Frontiers in Immunology, 10, 2276.
  5. Tanaka, M., Saka-Tanaka, M., Ochi, K., Fujieda, K., Sugiura, Y., Miyamoto, T., … & Aoe, S. (2020). C-type lectin Mincle mediates cell death–triggered inflammation in acute kidney injury. Journal of Experimental Medicine, 217(11).
  6. Becker, N., Störmann, P., Janicova, A., Köhler, K., Horst, K., Dunay, I. R., … & Relja, B. (2021). Club Cell Protein 16 Attenuates CD16brightCD62dim Immunosuppressive Neutrophils in Damaged Tissue upon Posttraumatic Sepsis-Induced Lung Injury. Journal of Immunology Research, 2021.
  7. Günther, H. S., Henne, S., Oehlmann, J., Urban, J., Pleizier, D., Renevier, N., … & Wülfing, C. (2021). GFAP and desmin expression in lymphatic tissues leads to difficulties in distinguishing between glial and stromal cells. Scientific Reports, 11(1), 1-14.
  8. Kawada, M., Yokoi, H., Kimura, T., Matsumoto, Y., Sakurai, H., Matsumoto, K., … & Saito, K. (2021). Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis. Allergology International.
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Minute™ Plasma Membrane/Protein Isolation and Cell Fractionation Kit 

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Minute™ Plasma Membrane/Protein Isolation and Cell Fractionation Kit (50 Preps)

Cat #: SM-005

Description

Free of detergents and EDTA, the Minute™ kit is a next-generation solution for native plasma membrane (PM) isolation and cell fractionation. It offers exceptional convenience and consistency by eliminating the variability often seen with traditional methods such as homogenization, density gradient centrifugation, and two-phase partitioning.

How it works: cells/tissues are first sensitized by buffer A before passing through the proprietary filter in a zigzag manner when high-speed centrifugal force is applied, resulting in a cell lysate containing ruptured cell membranes and intact nuclei. As a result, nuclear contamination is virtually eliminated. PM is further separated from the cell lysate (a mixture of crude membranes, intact nuclei, cytosol proteins, and organelles) by subsequent differential and density centrifugation with a regular tabletop microcentrifuge. 5 distinct cell fractions (total membrane, PM, cytosol, nucleus, and organelles) can be obtained. The procedure can be completed in less than 45 minutes.

Kit includes:

Items

Quantity

Buffer A

25 ml

Buffer B

10 ml

Protein Extraction Filter Cartridges

50 units

Collection Tubes with Caps

50 units

Plastic Rods

2 units

Tissue Dissociation Beads

10 grams


Figure 1. A. SDS-PAGE profiles of total cell lysate (TC) and isolated plasma membrane proteins (PM) from human and rat cultured cells. Lane 1, Human lung cancer cell A549 total cell lysate; Lane 2, Isolated PM of A549; Lane 3, PM of rat REL-6TN cells; Lane 4, Total cell lysate of rat REL-6TN cells.

B. Western blotting: Proteins in A were transferred to nitrocellulose membrane and probed with anti-Na/K ATPase alpha1, a plasma membrane marker (Upstate, Clone 464.6), and anti-lamin B1, a nuclear envelope marker (ab16048, Abcam Cambridge, MA). The specific protein bands were visualized by a color metric substrate Opti-4CN (Bio-RAD).

C. Densitometry measurement of Na/K ATPase alpha1 signals in B (TC vs. PM). Total cell lysates were extracted by Minute Denaturing Total Protein Extraction Kit (SD-001, Invent Biotechnologies, Eden Prairie, MN).

Figure 2 A. SDS-PAGE profiles of isolated total membrane proteins from mouse tissues (T=Total Cell Lysate, C=Cytosol Fraction, M= Total Membrane Fraction)

B. Proteins shown in A were transferred to a nitrocellulose membrane and probed with rabbit-anti mouse pan-cadherin (ab6529, Abcam, Cambridge, MA), and anti-actin by Western blotting. The specific protein bands were visualized by a color metric substrate Opti-4CN (Bio-RAD). Signals of pan-cadherin (a 130 kDa plasma membrane marker) were significantly enhanced in total membrane protein fractions. Total cell lysates were extracted by Minute™ Denaturing Total Protein Extraction Kit (SD-001, Invent Biotechnologies, Plymouth, MN).

Figure 3. A. SDS-PAGE profiles of total membrane protein fraction (TM) and isolated plasma membrane protein fraction from mouse tissues. 

B. Western blotting of proteins in A were transferred to nitrocellulose membrane and probed with rabbit anti-cadherin (abcam, Cambridge, MA). The specific protein bands were visualized by a color metric substrate Opti-4CN (Bio-RAD). 

C. Densitometry measurement of cadherin signals in B (TM vs. PM).

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Minute™ Detergent-Free Plant Protein Extraction Kit

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Minute™ Detergent-Free Plant Protein Extraction Kit (50 Preps)

Cat #: SN-010

Description

Minute™ Detergent-Free Plant Protein Extraction Kit is composed of optimized protein extraction buffer and protein extraction filter cartridges with 2.0 ml collection tubes. The kit is designed to rapidly extract detergent-free total proteins from plant tissues. The protein extraction buffer does not contain any detergent or organic solvent.

The extraction volume can be as small as 50 μl and as large as 500 μl using the protein extraction filter cartridges. Detergent-free total proteins can be extracted from very small plant samples (leaves, seeds, soft stems, etc.) in less than 8 minutes with a high yield (1-6 mg/ml).

Kit includes:

Items

Quantity

DF-Lysis Buffer (with 5g beads)

25 ml

Protein Extraction Filter Cartridges

50 units

Collection Tubes with Caps

50 units

Plastic Rods

2 units


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Minute™ Total Protein Extraction Kit for Plant Tissues

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Minute™ Total Protein Extraction Kit for Plant Tissues (50 Preps)

Cat #: SD-008/SN-009

Description

The MinuteTM total protein extraction kit for plant tissues comprises an optimized protein extraction buffer and protein extraction filter cartridges accompanied by 2.0 ml collection tubes. It aims to efficiently extract denatured or native proteins from various plant tissues, including leaves, seeds, soft stems, and roots. Notably, the protein profiles obtained through denaturing and native cell lysis buffers differ. Therefore, depending on the specific application, one buffer may outperform the other. This kit offers both denaturing and native cell lysis buffers, allowing users to evaluate and select the most suitable option for their particular needs. Utilizing the protein extraction filter cartridges makes it possible to extract total plant soluble proteins from 50-200 mg of plant tissue within 5-8 minutes while achieving a high protein yield ranging from 2-8 mg/ml. This extraction method is useful for SDS-PAGE, immunoblotting, ELISA, IP, and enzyme assays. These extracted proteins can also be excellent starting materials for small-scale protein purification using column chromatography techniques.

Kit includes:

Items

Quantity

Denaturing Lysis Buffer

25 ml

Native Lysis Buffer

25 ml

Potein Extraction Filter Cartridges

50 units

Collection Tubes with Caps

50 units

Plastic Rods

2 units

 

References (340)
  1. Crystal R. et al. (2013). Expression profiling of four defense-related buffalograss transcripts in response to chinch bug (hemiptera: Blissidae) feeding. Journal of Economic Entomology. 106 (6):2568-2576.
  2. Weber, C. F. (2017). Microgreen Farming and Nutrition: A Discovery-Based Laboratory Module to Cultivate Biological and Information Literacy in Undergraduates. The American Biology Teacher, 79(5), 375-386.
  3. Abdulazeez, M., Abubakar, S. M., & Mu’azzam, J. M. (2017). Isolation and Characterization of a Potential Angiotensin-Converting Enzyme Inhibitory Peptide from the Leaves of Leptadenia hastata (Asclepiadaceae). Malaysian Journal of Applied Sciences, 2(1), 35-47.
  4. Shi, Z., Jiang, Y., Han, X., Liu, X., Cao, R., Qi, M., … & Li, T. (2017). SlPIN1 regulates auxin efflux to affect flower abscission process. Scientific Reports, 7(1), 14919.
  5. Ohno, S., Hori, W., Hosokawa, M., Tatsuzawa, F., & Doi, M. (2018). Post-transcriptional silencing of chalcone synthase is involved in phenotypic lability in petals and leaves of bicolor dahlia (Dahlia variabilis)‘Yuino’. Planta, 247(2), 413-428.
  6. Jesus, F. G., Marchi-Werle, L., Fischer, H. D., Posadas, L. G., Graef, G. L., & Heng-Moss, T. Documenting Resistance and Physiological Changes in Soybean Challenged by Aphis glycines Matsumura (Hemiptera: Aphididae). Neotropical Entomology, 1-8.
  7. Lv, Z., Huang, Y., Ma, B., Xiang, Z., & He, N. (2018). LysM1 in MmLYK2 is a motif required for the interaction of MmLYP1 and MmLYK2 in the chitin signaling. Plant Cell Reports, 1-12.
  8. Marchi-Werle, L., Fischer, H. D., Graef, G., Hunt, T. E., & Heng-Moss, T. M. (2018). Characterization and Identification of Methods for Phenotyping Soybean Populations With Tolerance to the Soybean Aphid (Hemiptera: Aphididae). Journal of Economic Entomology. toy177, https://doi.org/10.1093/jee/toy177
  9. Wang, J., Xia, H., Zhao, S. Z., Hou, L., Zhao, C. Z., Ma, C. L., … & Li, P. C. (2018). A role of GUNs-Involved retrograde signaling in regulating Acetyl-CoA carboxylase 2 in Arabidopsis. Biochemical and Biophysical Research Communications.
  10. Chapman, K. M., Marchi-Werle, L., Hunt, T. E., Heng-Moss, T. M., & Louis, J. (2018). Abscisic and Jasmonic Acids Contribute to Soybean Tolerance to the Soybean Aphid (Aphis glycines Matsumura). Scientific Reports, 8(1), 15148.
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Minute™ Detergent-Free Protein Extraction Kit for Animal Tissues and Cultured Cells

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Minute™ Detergent-Free Protein Extraction Kit for Animal Tissues and Cultured Cells (50 Preps)

Cat #: SN-006

Description

Minute™ Detergent-Free Total Protein Extraction Kit is composed of optimized cell lysis buffer and protein extraction filter cartridges with 2.0 ml collection tubes. The kit is designed to quickly extract detergent-free total proteins from cultured cells (insect/mammalian/other cultured cells) and animal tissues. The protein extraction buffers do not contain any detergent and EDTA. The extraction volume can be as small as 20 μl and as large as 500 μl with the use of the protein extraction filter cartridges. When available starting material is a limiting factor, this can be quite useful. Detergent-free total proteins can be extracted from cultured cells/tissues in less than 5 minutes with high yield (1-5 mg/ml).

Kit includes:

Items

Quantity

Buffer A

15 ml

Buffer B

15 ml

Protein Extraction Filter Cartridges

50 units

Collection Tubes with Cap

50 units

Plastic Rods

2 units

Figure 1. Comparison of protein profiles of different animal tissues extracted by denaturing lysis buffer and detergent-free extraction buffer. Lane 1: Drosophila cultured cells (SL2); Lane 2: Adult Drosophila flies; Lane 3: Gold fish muscle; Lane 4: Mouse liver. Extracted proteins were seperated in 10% SDS-PAGE and stained with Comassie blue.

References (8)
    1. Seo, J-H. et al (2014). Directing stem cell differenciation by changing the molecular mobility of supramolecular surfaces. Advanced Healthcare Materials. DOI: 10.1002/adhm.201400173
    2. M. Takada, S. Chiba et al. ((2015). Inflammatory responses to neutral fat and fatty acids in multiple organs in a rat model of fat embolism syndrome, Forensic Science International http://dx.doi.org/10.1016/j.forsciint.2015.07.011
    3. Seo J. et al. (2016). Dynamic polyrotaxane-coated surface for effective differentiation of mouse induced pluripotent stem cells into cardiomyocytes. RSC Advances. DOI: 10.1039/C6RA03967G.
    4. Xu, Wenteng, et al. (2016).”Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination.” Gene.
    5. Yamamoto, H., Hayano, S., Okuno, Y., Onoda, A., Kato, K., Nagai, N., … & Kato, T. (2020). Phosphorylated proteome analysis of a novel germline ABL1 mutation causing an autosomal dominant syndrome with ventricular septal defect. International Journal of Cardiology.
    6. Pietras, P., Leśniczak-Staszak, M., Kasprzak, A., Andrzejewska, M., Jopek, K., Sowiński, M., … & Szaflarski, W. (2021). MVP Expression Facilitates Tumor Cell Proliferation and Migration Supporting the Metastasis of Colorectal Cancer Cells. International Journal of Molecular Sciences, 22(22), 12121.
    7. Minerva, D., Othman, N. L., Nakazawa, T., Ito, Y., Yoshida, M., Goto, A., & Suzuki, T. (2022). A New Chemotactic Mechanism Governs Long-Range Angiogenesis Induced by Patching an Arterial Graft into a Vein. International Journal of Molecular Sciences, 23(19), 11208.
    8. Leśniczak-Staszak, M., Pietras, P., Ruciński, M., Johnston, R., Sowiński, M., Andrzejewska, M., … & Szaflarski, W. (2024). Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention. Journal of Cell Science, 137(12).
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Minute™ Total Protein Extraction Kit for Animal Cultured Cells and Tissues

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Minute™ Total Protein Extraction Kit for Animal Cultured Cells and Tissues (50 Preps)

Cat #: SD-001/SN-002

Description

The Minute™ kit is a next-generation, spin column–based system designed for rapid and efficient total protein extraction from animal cultured cells and tissues. Unlike conventional solution-based methods, such as RIPA buffer, which often result in protein loss and altered profiles, this patented technology preserves protein integrity and yields reproducible results.

With enhanced lysis buffers and a streamlined protocol, proteins can be extracted in just 1–5 minutes at concentrations ranging from 1–8 mg/ml. The system supports both denaturing and native conditions, allowing users to choose based on downstream applications. Minimal extraction volume (as low as 20 µl) makes it ideal when starting material is limited.

This kit offers one of the fastest protocols available for total protein preparation and can be readily adapted for high-throughput workflows. The extracted proteins are also suitable as starting material for small-scale purification using column chromatography.

-RIPA Buffer – The Potential Troubles

-Pitfalls of Protein Extraction by RIPA

-A Technical Breakthrough for Protein Sample Preparation

Selected references related to protein loss by RIPA buffer:

  1. Bai, B., and Laiho, M. (2012) Proteomics. 12:3044-3048
  2. Mukhopadhyay, C. et al. (2016) PNAS 5:8228-8237
  3. Li, Q. (2016) Biotechniques. 61:327
  4. Ngoka, L. CM. (2008) Proteome Science. 6:30

Protein profiles of animal tissues extracted by denaturing lysis buffer and native lysis buffer. Extracted proteins were separated in 10% SDS-PAGE and stained with coomassie blue. Lane 1, drosophila larvae; lane 2, drosophila pupa; 3, adult drosophila; 4, goldfish muscle and 5, mouse liver. 

Kit includes:

Items Quantity
Denaturing Cell Lysis Buffer 25 ml
Native Cell Lysis Buffer 25 ml
Protein Extraction Filter Cartridges 50 units
Collection Tubes with Caps 50 units
Plastic Rods 2 units

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