1. נוהל quantitation DNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך על המכשיר. </li><li style=";text-align:right;direction:rtl"> פתח את תוכנת BioSpec ננו משולחן העבודה במחשב. </li><li style=";text-align:right;direction:rtl"> לחץ על הכרטיסייה פשוט חומצה dsDNA גרעין (איור 1). </li><li style=";text-align:right;direction:rtl"> התוכנה יהיה ליצור תקשורת עם המכשיר ולהפעיל רצף האתחול. זה מוודא כי המכשיר מתפקד בהתאם למפרט. </li><li style=";text-align:right;direction:rtl"> בחר 0.7 או 0.2 מ&quot;מ על המחוון pathlength. זה בוחר את pathlength וכל החישובים מבוססים על pathlength שנבחר. </li><li style=";text-align:right;direction:rtl"> הבא BioSpec ננו ההתקנה לחץ על הגדרת-Autowiping תוכנה. בחר 1 או 2. </li><li style=";text-align:right;direction:rtl"> פיפטה 2 μL (0.7 מ&quot;מ) H 2 O או חיץ על הכן. </li><li style=";text-align:right;direction:rtl"> לחץ ריק או F6 למדידת ריק. בכל פעם pathlength חדשה נבחרה יש צורך להקליט ריק. </li><li style=";text-align:right;direction:rtl"> פיפטה 2 μL של המדגם על הכן. </li><li style=";text-align:right;direction:rtl"> לחץ על התחל בתוכנה או ללחוץ על הכפתור להתחיל על המכשיר. החלון העליון מהלכים ליצור קשר עם רביב המדגם. מהבהב קסנון ניתן לצפות. </li><li style=";text-align:right;direction:rtl"> חזור על מדגם זהה 3 פעמים כדי לקבוע reproducibility. </li><li style=";text-align:right;direction:rtl"> כל הנתונים נרשם נשמר כקובץ. ניצן. </li><li style=";text-align:right;direction:rtl"> כדי לשמור כקובץ. Pdf או. קובץ CSV לחץ על שמור PDF / csv או F9. תוצאות ניתן גם לשמור כקובץ csv. באמצעות בחירה בלשונית המתאימה לשמור. </li></ol> 2. פרוטוקול quantitation חלבון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר quantitation הכרטיסייה חלבון (איור 2). </li><li style=";text-align:right;direction:rtl"> הזן את e280 מקדם הכחדה, אם הוא ידוע. </li><li style=";text-align:right;direction:rtl"> אם e280 אינו ידוע לחשב אותו באמצעות e280Calc. </li><li style=";text-align:right;direction:rtl"> בחר 0.7 או 0.2 מ&quot;מ על המחוון pathlength. </li><li style=";text-align:right;direction:rtl"> לחץ על Setup-Autowiping ההתקנה. בחר 2 ומעלה. </li><li style=";text-align:right;direction:rtl"> פיפטה 3 μL (0.2 מ&quot;מ) H 2 O או חיץ על הכן. </li><li style=";text-align:right;direction:rtl"> לחץ ריק או F6 למדידת ריק. </li><li style=";text-align:right;direction:rtl"> פיפטה 3 μL מדגם חלבון על הכן. </li><li style=";text-align:right;direction:rtl"> לחץ על התחל בתוכנה או ללחוץ על הכפתור להתחיל על המכשיר. </li><li style=";text-align:right;direction:rtl"> חזור על מדגם זהה 3 פעמים כדי לקבוע reproducibility. </li><li style=";text-align:right;direction:rtl"> כל הנתונים נרשם נשמר כקובץ. ניצן </li><li style=";text-align:right;direction:rtl"> כדי לשמור כקובץ. Pdf או. קובץ CSV לחץ על שמור PDF / csv או F9. </li></ol> 3. נציג תוצאות: 1. תוצאות עבור quantitation DNA איור 3 מציג את נתוני מדידות על דגימת DNA גדילי כפול טיפוסי. ריכוז ו OD 260/280 יחס מחושב מוצגים גם. איור 4 מכיל מבחן reproducibility תוצאות quantitation DNA. הנתונים איורים 3 ו -4 תוצאות מראה ולייצר מאוד כפי שעולה סטיית תקן נמוכה יחסית של 0.26%. הערה 1: אי דיוק quantitation DNA יכול לנבוע נוכחות של רכיבים חיץ אשר עשוי לספוג באזור UV. זהו גם צורך להשתמש בפתרון מדגם הומוגני. ככל שכמות מדגם pipetted להיות רק 1-2 pipetting μL, נזהר כרכים מדויק ללא הקדמה של בועות בכל הנדרש. 2. תוצאות עבור quantitation חלבון איור 5 מייצג ריכוז חלבון ניתוח BSA. ריכוז מיקרוגרם / מ&quot;ל ​​חלבון מבוססת על ערך e280. איור 6 מציג ניתוח שחזור. ברור מן סטיית התקן היחסית של 0.51% כי BioSpec ננו ריכוז אמצעים בדיוק - לא רק דנ&quot;א אלא גם חלבונים. הערה 2: גורמים המשפיעים על ריכוז חלבון קביעת ריכוז מדויק של חלבון אפשרית כאשר יש תופעות מינימום מחוסר יציבות הנגרמת על ידי ה-pH, מאגרים, טמפרטורה או תוספים. חלבונים מטוהרים רוב עובר שלבים שונים של עיבוד, ולכן ייתכן כי החלבון עשוי להכיל חומרים פעילי שטח או תוספים אחרים שעשויים להפריע ליצירת טיפה. לקבלת דוגמיות כאלה, באמצעות 5 מ&quot;מ pathlength תא קוורץ מומלץ מאוד. <img src="/files/ftp_upload/2699/2699fig1.jpg" alt="איור 1" /> 1. איור אנליטי מבחר חלון quantitation DNA גדילי כפול <img src="/files/ftp_upload/2699/2699fig2.jpg" alt="איור 2" /> 2. איור אנליטי מבחר חלון quantitation ללא חלבון הנקרא <img src="/files/ftp_upload/2699/2699fig3.jpg" alt="איור 3" /> איור 3. נציג נתונים עבור quantitation DNA <img src="/files/ftp_upload/2699/2699fig4.jpg" alt="איור 4" /> איור 4. שחזור נתונים quantitation DNA <img src="/files/ftp_upload/2699/2699fig5.jpg" alt="איור 5" /> איור 5. נציג נתונים עבור quantitation חלבון <img src="/files/ftp_upload/2699/2699fig6.jpg"alt = &quot;איור 6&quot; /&gt; איור 6. שחזור נתונים עבור quantitation חלבון

<ol>
	<li>Aisubel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Short Protocols in Molecular Biology. , (1999).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+predict+molar+absorption+coefficient+of+a+protein.">How to measure and predict molar absorption coefficient of a protein.</a> Protein Science. 4, 2411-2423 .</li></ol>

Suja Sukumaran, PhD הוא עובד של Shimadzu שעושה את המכשיר בהשתתפות במאמר זה.

BioSpec-nano תוכנן כדי לפתור את הבעיות הנוכחיות הטמון שיטות quantitation DNA, כגון תוצאות לא עקביות ועל עבודת כפיים מייגעת. מדידת כל לוקח רק 3 שניות, מה שמאפשר ביולוג מולקולרי מנוסה לנתח דגימות מרובות על 3-6 תוך דקה. תכונת מגב ייחודי לא רק משפרת את התפוקה של המדידה, היא גם מבטלת את הצורך בניקוי ידני של כנים, וכתוצאה מכך שריד זניח בין המדידות. יתר על כן, רק 2 בדיקות באמצעות מגבונים עבור דגימות DNA המכיל 1000 ng / μL, ציין שריד פחות מ 2 ng / μL. הזמינות של תא pathlength 5 מ&quot;מ מספק אפשרות לנתח חומצות גרעין לדלל דגימות חלבון, מרחיב את השימוש במכשיר זה עבור יישומים אחרים.

<table><tbody><tr><td>BioSpec-nano</td><td>Shimadzu Corporation</td><td></td><td></td></tr><tr><td>Calf Thymus DNA</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

concentration determination of nucleic acids and proteins using the micro-volume bio-spec nano spectrophotometer

גרעין quantitation נהלים חומצה יש מתקדמות באופן משמעותי בשלושת העשורים האחרונים. יותר ויותר, ביולוגים מולקולריים דורשים ניתוח בקנה אחד קטן בנפח של דגימות חומצות גרעין עבור הניסויים שלהם. BioSpec ננו מספק פתרון אפשרי לבעיות לא מדויק, לא לשחזור תוצאות, הטמון שיטות quantitation הנוכחית DNA, באמצעות האופטיקה המקצועית גלאי PDA רגיש. BioSpec ננו יש גם פונקציונליות אוטומטיות כזו הרכבה, מדידה, ניקוי נעשים על ידי המכשיר, ובכך למנוע השמה משעמם, חוזר על עצמו, וגם לא עקבית של אלמנט סיב אופטי ניקוי ידני. במחקר זה, הנתונים שהוצגו על כימות של דנ&quot;א וחלבונים, כמו גם על שחזור מדידה ודיוק. מגע מדגם אוטומטי לסריקה מהירה מאפשר מדידה תוך שלוש שניות, וכתוצאה מכך תפוקה מעולה. ניתוח הנתונים מבוצע באמצעות תכונות מובנות של התוכנה. הנוסחה המשמשת לחישוב ריכוז ה-DNA הוא: לדוגמא ריכוז = DF ° (OD260-OD320) · NACF (1) איפה DF = דילול מדגם גורם ו NACF = גורם גרעין ריכוז החומצה. גורם גרעין ריכוז חומצה מוגדר בהתאם אנליטי שנבחר 1. ריכוז תוצאות חלבון ניתן לבטא מיקרוגרם / מ&quot;ל ​​או שומות / L ידי הזנת e280 וערכים משקל מולקולרי בהתאמה. כאשר ערכים שאריות של Tyr, TRP ו - ציסטאין (SS האג&quot;ח) מוזנים בכרטיסיה e280Calc, מקדם את הערכים הכחדה מחושבים e280 = 5,500 x (שאריות TRP) + 1490 x (שאריות Tyr) + 125 x (ציסטאין אס האג&quot;ח) . ערך e280 משמש תוכנה לחישוב ריכוז. בנוסף לקביעת ריכוז של חומצות גרעין וחלבונים, BioSpec ננו יכול לשמש ספקטרופוטומטר מיקרו נפח אולטרה עבור analytes רבים אחרים או ספקטרופוטומטר תקן באמצעות 5 pathlength תאים מ&quot;מ.

Watch this Scientific Journal Video about Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer at JoVE.com

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

תקשורת זה מובאים נתונים על דיוק ועל שחזור של ספקטרופוטומטר UV-VIS BioSpec ננו עבור dsDNA ו quantitation חלבון. אפילו עם כמויות קטנות במיוחד (1 עד 2 ליטר), שחזור מצוין, בעוד פונקציה מנגב אוטומטי משפר את התפוקה ואת התוצאות שריד מינימלי תוצאות מדויקות יותר.

קביעת ריכוז חומצות גרעין וחלבונים שימוש Micro בנפח Bio-spec ספקטרופוטומטר Nano

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

concentration-determination-nucleic-acids-proteins-using-micro-volume

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

39.0K Views.  Scientific Instruments. תקשורת זה מובאים נתונים על דיוק ועל שחזור של ספקטרופוטומטר UV-VIS BioSpec ננו עבור dsDNA ו quantitation חלבון. אפילו עם כמויות קטנות במיוחד (1 עד 2 ליטר), שחזור מצוין, בעוד פונקציה מנגב אוטומטי משפר את התפוקה ואת התוצאות שריד מינימלי תוצאות מדויקות יותר.

Video: Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

מבחני colorimetric מהירים לאופן איכותי להבחין RNA ו-DNA בדגימות biomolecular

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Chemistry

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

התקנים עבור כמותי Metalloproteomic ניתוח באמצעות גודל הרחקת ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

הערכת משלוח גן פולימרית חלקיקים על ידי Nanoparticle מעקב ניתוח וזרימת cytometry תפוקה גבוהה

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Bioengineering

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

ריכוז חלבון Microvolume קביעת באמצעות 2000c NanoDrop ספקטרופוטומטר

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

אפיון ספקטרוסקופי UV-Vis של ננו-חומרים במדיה מימית

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

Engineering

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

ה-DNA צינוריות כמו כלי רב-תכליתי ללמוד פולימרים Semiflexible

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

כימות dsDNA באמצעות ה-F-7000 Hitachi Fluorescence ספקטרופוטומטר ו PicoGreen דיי

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop Microvolume quantitation של חומצות גרעין

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

ניתוח מעקב חלקיקים לכימות וקביעת גודל של שלל חוץ-תאי

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Photometric Protein Determination

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is unknown.
This video describes the basics of photometric protein determination and introduces the Bradford Assay and the Lowry Method. The procedure in the video will cover a typical Bradford assay. Applications covered include direct measurement of very small volumes of nucleic acids to characterize concentration and purity, determination of coupling efficiency of a biomimetic material, and another variation of photometric protein determination using Remazol dye.
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	Determining the concentration of a protein in samples is a fundamental step in many biochemical assays. Photometric determination can be done with small sample sizes. The more a sample contains light-absorbing substances, the less the light will transmit through it. This provides a quantitative measurement of the absorbing substances. These concepts are so fundamental to science that the articles that introduced two of the techniques are in the three most cited papers of all time. This video will show the concepts behind some of the most common photometric protein determination techniques, how they are performed, and how the gathered data is analyzed.
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	Photometric protein determination is based on the relationship between concentration and light absorbency. This is known as the Beer-Lambert Law, which states that the concentration of a light-absorbing species is proportional to its absorbance.
	This principle underlies all photometric protein determination methods. 
	For direct absorption analysis, the absorbance values of unaltered protein samples are measured. Because of their aromatic side chains, tryptophan and tyrosine residues give the highest absorbance readings at a wavelength of 280 nm.
	However, these amino acids-which are two of the least frequently found in proteins-are present in different amounts in every protein, so each determination is unique. To overcome this limitation, more complex assays-that are not dependent on these amino acids-were developed.
	One example is the Bradford Assay, where colored dye is added to the sample. The dye, known as Coomassie Blue, responds proportionally-the more protein present, the more binding events with the dye.
	Then, protein concentration is determined by measuring the absorbance of the bound Coomassie Blue dye, which absorbs light at 594 nm. However, the Bradford assay is linear over a short range of concentrations, so dilutions are often required before analysis.
	The Lowry Method combines the Biuret reagent, an alkaline solution of copper ions that react with peptide bonds, and the Folin-Cioc&#226;lteu reagent, which oxidizes aromatic protein residues. The resulting color change of the sample is proportional to the protein concentration.
	The absorbance of the reduced Folin reagent can be determined at 750 nm. Like direct absorption, each protein has a unique response, and must be calibrated for the protein of interest. Now that we've reviewed the basic principles behind some of the most common assays, let's look at how direct absorption and the Bradford assay are performed.
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	To begin a direct absorption analysis, the spectrophotometer is calibrated with a blank to determine zero absorbance. Standard solutions are prepared for use in creating the calibration curve. Then, an aliquot of the first standard is added to a cuvette, and placed into the spectrophotometer.
	The absorbance value at 280 nm is then recorded. This process is repeated for each standard, using a clean cuvette for each run. Once complete, a calibration curve is created by plotting the absorbance versus concentration. The slope of this line is the molar attenuation coefficient, which relates absorbance to concentration.
	Next, the unknown sample is added to a cuvette, and the absorbance value is recorded. As the data analysis for the different photometric determination methods is similar, we will cover that after we look at the Bradford assay.
	Here, the Bradford protein assay is performed with a BSA standard on a 96-well plate. To begin, BSA stock solutions are prepared.
	The unknown solutions are diluted with deionized water to ensure that the concentrations are within the assay's range. Depending on the kit, the Coomassie dye may also require dilution. Then, the calibration curve is set up by adding the BSA standards to the 96-well plate.
	Deionized water is added to reach the needed concentration to generate a standard curve. The unknown sample should be added to the plate in triplicates to ensure an accurate measurement is taken. Coomassie dye is next added to each well, mixing with the pipette.
	Deionized water is added to an empty well as a blank, to measure the absorbance. After waiting 5 min for the dye to bind, the absorbance is measured in a plate-reader at 590 nm.
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	Now that we've performed a few assays, let's look at how to analyze the data. Each photometric protein determination method is based on the Beer-Lambert Law.
	The measured absorbance of the standards is used to create a calibration curve, which is then used to determine the concentration of unknown samples. This curve can be manually plotted, though newer spectrophotometric tools will create the calibration curve once all standards have been measured. These systems will also calculate protein concentration as unknown samples are analyzed.
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	Now that we've reviewed how to analyze photometric protein determination data, let's look at some of the ways these procedures are utilized.
	The principles of photometric protein determination can also be used to directly measure nucleic acid concentration. The nanodrop spectrophotometer accepts samples of very small volume onto an optically active pedestal. The absorbance is then measured, and the system automatically determines the nucleic acid concentration. Because proteins and other sources can interfere with measurements, sample purity is determined by analyzing the 260 to 280 nm and 260 to 230 nm absorbance ratios. Pure nucleic acids typically yield ratios of approximately 1.8 and approximately 2.0 for DNA and RNA, respectively.
	Photometric protein determination can also be used in the production of biomimetic materials, which are inspired from nature to elicit specific cellular responses. Recombinant adhesins are bound to polystyrene beads to simulate bacterial attachment to host cells. The Bradford assay is used to determine the coupling efficiency of the recombinant adhesion to the beads in the production of the biomimetic material.
	Alternative photometric protein assays can be used in the detection and characterization of protein antimicrobials. Remazol brilliant blue R dye is covalently bonded to heat-killed bacteria. The protein antimicrobial is incubated in the dyed solution. Then, the sample is centrifuged, and the absorbance of the supernatant at 595 nm is measured using a microplate spectrophotometer. Increased absorbance, by the soluble dye released into the supernatant from the labeled bacteria, is a quantitative measurement of enzymatic activity.
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	You've just watched JoVE's video on photometric protein determination. This video described the underlying principles of photometric determination, went over general procedures for some common assays, and covered some new advances in techniques. Thanks for watching!
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Photometric Protein Determination: Bradford Assay

קביעת חלבונים פוטומטריים

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more ...