Hielscher Ultrasonics
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Bizga qo'ng'iroq qiling: +49 3328 437-420
Bizga xat yuboring: info@hielscher.com

To'qimalar va hujayra madaniyatlaridan ultratovushli oqsil ekstraktsiyasi

  • Protein extraction is an essential sample preparation step in proteomics.
  • Proteins can be extracted from plant and animal tissue, yeasts and microorganisms.
  • Sonication is a reliable, efficient protein extraction method giving high protein yields within a short extraction time.

Protein Extraction from Tissues and Cells

Protein extraction from tissues and cultured cells is an essential sample preparation step which is performed during many biochemical and analytical techniques such as ELISA, PAGE, Western blotting, mass spectrometry, or protein purification. Ultrasonic cell disruption, lysis and extraction is a precisely controllable, non-thermal technique to ensure high yields of proteins.

Ma'lumot so'rovi







Probe-type ultrasonicators such as the UP200St are reliable tissue homogenizers and widely used for sample preparation in genetic and proteomic research.

Protein extraction from cells with the ultrasonic probe UP200St

Advantages of Ultrasonic Lysis and Protein Extraction
 

  • Rapid
  • yuqori hosildorlik
  • Yuqori samarali
  • Precise control over parameters
  • takrorlanadigan natijalar
  • chiziqli miqyoslilik

General Instructions For Ultrasonic Lysis and Protein Extraction

  • Temperature control: To ensure a high protein yield without thermal denaturation, temperature during extraction must be controlled. Hielscher’s state-of-art ultrasonic homogenizersalso called ultrasonic disintegrator or ultrasonifierare precisely controllable. They come with a plugable temperature sensor. In the setting options of the ultrasonic homogenizer, a maximum temperature can be set. When this temperature max is reached, the ultrasonicator automatically stops until the sample has cooled down.
  • Buffer: The choise of a suitable buffer and the right volume of buffer varies from tissue to tissue and must be figured-out by trial-and-error testing.
  • Isolation / purification: Protein lysates can contain an excess of biomolecules such as DNA or carbohydrates, which may be removed by protein precipitation (deoxycholate-trichloroacetic acid) or buffer exchange.

Chittapalo and Noomhorm (2009) reported in their study that protein yield increased using sonication and that the ultrasonic tissue homogenization and lysis process can enhance existing extraction processes significantlyenabling for new commercial extraction opportunities.
 

Ultrasonic CupHorn for the simultaneous sample preparation of multiple samples under same conditions for protein isolation and DNA fragmentation.

Ultrasonik kubok for the simultaneous, highly efficacious sample prep of multiple samples under same conditions for protein isolation and DNA fragmentation.

Protein Extraction from Animal Tissue

Ultrasonicator UP100H ko'pincha hujayra madaniyati plitalarining namunalarini tayyorlash uchun ishlatiladigan laboratoriya gomogenizatoridir.For the preparation of whole-sized tissue (e.g. kidney, heart, lung, muscle etc.), the tissue should be dissected into very small pieces with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases (e.g. lysis buffer such as RIPA or hypotonic lysis buffer containing protease and phosphatase inhibitor cocktail). After dissecting, the sample is immersed into liquid nitrogen for snap freeze. The sample can be stored at -80°C for later use or be keept on ice for immediate homogenization. Immediately before ultrasonic extraction, ice cold lysis buffer (with protease inhibitors DTT, leupeptin and aprotinin) is rapidly added into the sample tube (per ~10 mg of tissue approx. ~600 μL of buffer are recommended). Approx. 20-60mg of tissue is recommended per sample tube.
Ultrasonic homogenization, lysis and extraction is performed with an ultrasonic homogenizer such as the UP100H or UP200Ht, equipped with an micro-tip sonotrode. Sonication duration is 60-90 sec. at an ultrasonic cycle mode of 15 sec. sonication and 10 sec. resting time. The sample should be kept in ice all the time.
After ultrasonic homogenization / extraction, the lysate is centrifuged at 27,000g for approx. 20 min. Afterwards the supernatent is collected, so that the protein concentration can be determined by a protein assay such as Pierce protein assay BCA.

Protein Extraction from Blood Serum

For a homogeneous mixture of the serum and the phosphate buffer, the sample is vortexed first before ultrasonic cell lysis. For the ultrasonic lysis, the sample is sonicated with an ultrasonic lab homogenizer such as the UP100H for 8 cycles at 20% amplitude, for cycles of each 5 seconds on and 15 seconds off. Protein extraction is carried out by sonicating in cycles (pulsation mode) and by placing the sample on ice so that an overheating and thermal degradation of the sample is avoided. Since serum contains a large amount of high molecular weight proteins (such as albumin, α1-antitrypsin, transferrin, haptoglobulin, immunoglobulin G and immunoglobulin A), which interfere with the separation of low molecular weight proteins during IEF, it is recommended to deplete them from the serum using a depletion column.

Protein Extraction from Plant Tissue

Fresh, soft plant tissue, e.g. moss etc., can be easily disrupted by simply placing the chopped sample material in lysis buffer for sonication. Tough, ligenous plant tissues, such as seeds, fir needles etc., should be ground dry. Some hard, woody plant materials must be frozen and ground in liquid nitrogen before extracted by sonication. For plant cell culture suspensions, an ultrasonic treatment between 30 and 150 seconds in a lysis buffer is mostly sufficient. Tougher material such as pumpkin seeds require a more intense sonication as described below.
 

Ushbu qo'llanmada laboratoriyalarda liziz, hujayra buzilishi, oqsil izolyatsiyasi, DNK va RNK parchalanishi, tahlil va tadqiqotlar kabi namuna tayyorlash vazifalaringiz uchun qaysi turdagi sonikator eng yaxshi ekanligini tushuntiradi. Ilovangiz, namuna hajmi, namuna raqami va o'tkazish qobiliyati uchun ideal sonikator turini tanlang. Hielscher Ultrasonics siz uchun ideal ultratovushli homogenizatorga ega!

Fan va tahlilda hujayra buzilishi va oqsil olish uchun mukammal sonikatorni qanday topish mumkin

Video eskizi

 

Protocol for Ultrasonic Extraction of Albumin from Pumpkin Seeds

Ultrasonic tissue homogenizer UP400St with S24d40 - for protein extraction from plant and animal tissue (Click to enlarge!)For the ultrasonic protein extraction of albumin from fine-ground pumpkin seed powder, 10 g of defatted pumpkin seed powder and 100 mL of deionised water as solvent are added in a 250mL glass beaker. The protein extraction consists in two steps: First, the sample is sonicated with a probe-type ultrasonicator UP400St (400W, 24kHz) equipped with sonotrode S24d7. The glass beaker is placed in a cold water bath during the ultrasonic homogenization. The pluggable temperature sensor and temperature control settings of the ultrasonicator UP400St ensure that the sample temperature is always kept below 30°C. By the precise temperature control during sonication, a denaturation of albumin is avoided. Secondly, extraction was performed with a mixer at 200 rpm speed and at 30°C. Afterwards the beaker is transferred into a thermostatic shaker. Globulin is removed via dialysis with distilled water. After the globulin removal, the protein extract can be sampled for determination of the albumin profile and is subsequently adjusted to pI=3.0 using 0.1 M HCl for albumin coagulation. The solid phase is separated by centrifugation at 5000g, 20°C and redissolved in deionised water. Albumin coagulation is performed twice to increase the protein ratio in the albumin concentrate.

Ultrasonic alkaline protein extraction for the preparation of protein concentrate from rice bran shows that the ultrasonic treatment results in higher protein yield in a significantly shorter extraction timecompared to conventional extraction methods.

Ushbu videoklip Hielscher ultratovushli gomogenizator UP100H, laboratoriyalarda namuna tayyorlash uchun keng qo'llaniladigan ultratovush qurilmasini ko'rsatadi.

Ultrasonik gomogenizator UP100H

Video eskizi

Sample Preparation Protocol for Functional iNOS Enzyme

To obtain fully functional iNOS enzyme (e.g. for drug screening), the following protocol is recommended: The cell suspension should be placed on ice and is sonicated with an UP100H at 10µm amplitude at cycle mode of 5 sec. sonication and 25 sec. rest on ice. The procedure should be repeated approx. 3 times. The resting time in between sonication cycles reduces the temperature rise and will therefore reduce the risk of denaturation.

Ultrasonic Protein Solubilization

Sonication may accelerate the protein solubilization process, which usually requires several hours. In order not to overheat the sample and prevent protein degradation and modifications in solutions containing urea, the ultrasonic bursts should not last longer than few seconds.

VialTweeter - bu o'zaro ifloslanishsiz bir xil sharoitlarda 10 flakongacha sonikatsiya qilish uchun noyob ultratovush tizimi.

Yopiq flakonlarni sonikatsiya qilish uchun VialTweeter bilan UP200St

Video eskizi

Ultrasonikator UP200Ht mikrotipli S26d2 biologik namunalarning ultratovushli lizisi uchun.

Ultrasonicator UP200Ht with 2mm microtip S26d2 for the sonication of small samples.

Ultrasonic Equipment for Protein Extraction

Hielscher Ultrasonics offer a broad range of ultrasonic homogenizers for the disintegration of cells, tissues, bacteria, microorganisms, yeast, and spores.
Hielscher lab ultrasonicators are powerful and easy to operate. Built for 24/7 operation, they are designed as robust and efficient lab and bench-top devices. For all devices, the energy output and the amplitude can be precisely controlled. The broad range of accessories opens further setup options. The digital ultrasonicators such as the VialTweeter, UP200Ht, UP200St, and UP400St have an integrated temperature control and a built-in SD card for automatic data recording.
For the indirect, cross-contamination-free and simultaneous sonication of multiple samples, we offer the VialTweeter or the ultrasonic CupHorn.
The table below gives you an overview over our ultrasonicators for sample preparation, cell disruption and extraction. Click at the device type to get more information on each ultrasonic homogenizer. Our well-trained and long-time experiences technical staff will be glad to help you choosing the most suitable ultrasonicator for your sample preparation!
 

To'plam hajmi Oqim darajasi Tavsiya etilgan qurilmalar
up to 10 vials or tubes na VialTweeter
multiwell / microtiter plates na UIP400MTP
multiple tubes / vessels na chashka
1 dan 500 ml gacha 10 dan 200 ml / min UP100H
10 dan 1000 ml gacha 20-200 ml/min UP200Ht, UP200St
10 dan 2000 ml gacha 20 dan 400 ml / min UP400St

Depending on your application, material, and sample volume, we will recommend you the most suitable setup for your sample prep. Contact us today!

Biz bilan bog'lanish! / Bizdan so'rang!

Qo'shimcha ma'lumot olish uchun so'rang

Please use the form below to request additional information about ultrasonic homogenizers, applications in biotech and proteomics as well as prices. We will be glad to discuss your cell disruption and protein extraction process with you and to offer you an ultrasonicator meeting your requirements!









Iltimos, bizning Maxfiylik siyosati.




 

Videoda ultratovushli namuna tayyorlash tizimi UIP400MTP ko'rsatilgan, bu yuqori intensivlikdagi ultratovush yordamida har qanday standart ko'p quduqli plitalarning ishonchli namunalarini tayyorlash imkonini beradi. UIP400MTP ning odatiy qo'llanilishi hujayra lizisini, DNK, RNK va xromatinni kesish, shuningdek, oqsillarni ajratib olishni o'z ichiga oladi.

Ko'p quduqli plastinka sonikatsiyasi uchun ultrasonikator UIP400MTP

Video eskizi

Hielscher ultrasonikatorlari brauzer nazorati orqali masofadan boshqarilishi mumkin. Sonikatsiya parametrlari kuzatilishi va jarayon talablariga aniq sozlanishi mumkin.

Hielscher digital ultrasonicators feature a browser remote control and automatic data protocolling on an integrated SD-card.

To'qimalar namunalaridan oqsil olish uchun ultratovushli VialTweeter qurilmasi (kattalashtirish uchun bosing!)

VialTweeter for indirect sonication.



Bilishga arziydigan faktlar

proteomika

Proteomics is the research field that investigates proteins and the proteome. Proteins fullfil a vast array of vital functions within organisms. The proteome is the entire set of proteins expressed by a genome, cell, tissue, or organism at a certain time. The proteome varies with time and distinct requirements, or stresses, that a cell or organism undergoes. More specifically, it is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. The term is a blend of proteins and genome. Proteomics is the study of the proteome.

Protein

Proteins are are large biomolecules, so-called macromoleculeswhich are composed from one or more long chains of amino acid residues. Proteins are present in all organisms of both vegetal and animal origin and they are crucial for most of the biological functions. Since proteins contain a lot of biological information, they are extracted for analytical purpose, e.g. for proteomic research. The most important function performed by proteins include the catalysis of metabolic reactions, DNA replication, response to stimuli, and the transport of molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific three-dimensional structure that determines its activity. Proteins arebesides peptidesone of the key components of food. Therefore, proteomics is a powerful tool in food science to optimize processes, food safety and nutritional assessment.

Cloud Point Extraction

Cloud Point Extraction is a pre-analytical procedure to separate and pre-concetrate analytes. In combination with ultrasonication, cloud point extraction can be intensified making the process more efficient, faster, and environment-friendlier. With ultrasonication, cloud point extraction is a significantly more efficient method of analyte preparation. Read more about ultrasonically assisted cloud point extraction!

Gel Electrophoresis

Gel electrophoresis is the major method for separation and analysis of macromolecules such as DNA, RNA and proteins as well as their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent) and in biochemistry, molecular biology and proteomics to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

Cell Cultures

Cell culture is the controlled growing process by which cells are cultivated under controlled conditions. Cell culture conditions vary for each cell type. In general, the environment of a cell culture consists of a suitable vessel (e.g. Petri dish) with substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells need a surface or artificial substrate, whilst other cell cultures can be cultivated free floating in culture medium (suspension culture, cell suspension).
Mass cultures of animal cell lines are used in industrial production of viral vaccines and other biotechnologically derived products. Human stem cells are cultured to expand the number of cells and differentiate the cells into various somatic cell types for transplantation purposes.

Tissue Samples

The term tissue describes a cellular intermediate, where the cell material is on an organisational level between cells and a complete organ. In tissue, similar cells, from the same origin that together carry out a specific function, are assembled. By the functional grouping of multiple tissues, the complex structures of organs are formed.
Tissue is sampled for research in biology, histology/histopathology, parasitology, biochemistry, immunohistochemistry as well as to cultivate and extract DNA. It can be distinguished between animal (subdivision: mammal tissue) and plant tissue. Animal tissues are grouped into the four basic types of connective, muscle, nervous, and epithelial tissue. Plant tissue is subdivided into the following three tissue systems: the epidermis, the ground tissue, and the vascular tissue.
Tissue samples can be prepared from animal or plant parts, e.g. bone, muscle, leaves, etc.

Body Fluids

Blood, serum, plasma, cerebrospinal fluid, saliva and synovial fluid are body fluids, which offer a great source of diagnostically relevant information. Therefore, a sophisticated preparation of body fluid samples for analysis is important. The first difficulty is associated with the broad dynamic range of components present in body fluids.

Determination of protein concentration

Bradford assay, Lowry assay and bicinchoninic acid (BCA) assay are common assays to determine the concentration of proteins. Bovine serum albumin (BSA) is one of the most frequently used protein standard.

Lysis Buffer

Lysis buffer must be chosen in accordance to the cell material or tissue (tissue culture, plant, bacteria, fungi, etc.), and whether the cells are in a structure and the type of structure. A wide range of lysis buffers for extraction of proteins, membranes, and organelles are formulated with one or more detergents. The detergent is usually selected via trial-and-error tests orif availableaccording to an existing protein extraction protocol. The detergent must be compatible with the tissue source and the proteins. In general, the mildest detergent that works for a specific tissue / protein, is chosen in order to maintain the maximal functionality of the extract. Furthermore, in case of an extraction of membranes and organelles, a mild detergent keeps the membrane intact. Commonly used detergents in lysis buffers are mostly nonionic or zwitterionic, e.g. CHAPS, deoxycholate, Triton™ X-100, NP40, and Tween 20.
For instance, tissues such as brain, liver, intestine, kidney, spleen etc. can be simply buffered with RIPAhowever protease inhibitors and DTT (e.g. for gel electrophoresis) should be included.
Lysis buffer for skeletal muscle tissue (ice cold): 20 mM Tris (pH 7.8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 % Triton X-100, 10 % (w/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol supplemented with protease and phosphatase inhibitor cocktail
Table of common buffers and their pH range. In general, these buffers are normally used at concentrations of 20-50 mM.
 

Buffer pH range
Citric acid – NaOH 2.26.5
Sodium citratecitric acid 3.06.2
Sodium acetateacetic acid 3.65.6
Cacodylic acid sodium saltHCl 5.07.4
MES – NaOH 5.66.8
Sodium dihydrogen phosphatedisodium hydrogen phosphate 5.88.0
ImidazoleHCl 6.27.8
MOPS – KOH 6.67.8
Triethanolamine hydrochloride – NaOH 6.88.8
TrisHCl 7.09.0
HEPES – NaOH 7.28.2
Tricine – NaOH 7.68.6
Sodium tetraborateboric acid 7.69.2
Bicine – NaOH 7.78.9
Glycine – NaOH 8.610.6

Most buffers show a pH-dependence with temperature. This is especially true for Tris buffers. The pKa changes from 8.06 at 25°C to 8.85 at 0°C.
(pH and pKa of a buffer: pH measures the concentration of hydrogen ions in an aqueous solution. pKa (= acid dissociation constant) is a related, but more specific measure, in that it helps to predict how a molecule will act at a specific pH value.)

TRIzol

TRIzol is a chemical solution used to extract RNA/DNA/protein during the guanidinium thiocyanate-phenol-chloroform extraction. The use of ultrasonically assisted TRIzol extraction results in high DNA, RNA, and protein yields from the same sample and excels thereby other extraction methods.

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