Amerigo Scientific, a distribution company focused on providing critical products and services to the biomedical and life science research communities, now introduces HHD Broth to the research community. This new product can be used for the detection and differentiation of heterofermentative and homofermentative lactic acid bacteria.

Microbiology is a biological science that deals with the identification and characterization of microorganisms and their interactions with their surrounding environment. Microbiology consists of several sub-disciplines, including bacteriology, mycology, phycology, parasitology, and virology. The study of the ecology, biology, physiology, and genetic mechanisms of these diverse organisms provide important insights into other scientific fields, including medicine, agriculture, fermentation, and food processing.

Amerigo Scientific offers a wide range of chemicals and reagents, media, antimicrobial susceptibility testing solutions and food safety testing molecular solutions for public health, clinical laboratories, food companies, environmental screening and pharmaceutical laboratories. It now expands product portfolio and provides HHD BROTH (Catalog Number: M001088813BIO) for researchers, the medium that can be used in the detection and differentiation of heterofermentative and homofermentative lactic acid bacteria.

Lactic acid bacteria are widely distributed in nature and participate not only in production, but also in the spoilage of acidic foods. According to their metabolic properties, they can be divided into two physiological groups: heterofermentative bacteria and homofermentative bacteria. Heterofermentative lactic acid bacteria produce carbon dioxide, lactic acid, acetic acid, ethanol and mannitol from hexose; homofermentative lactic acid bacteria mainly produce lactic acid from hexose.

Homofermentative-Heterofermentative Differential (HHD) Broth now is available at Amerigo Scientific, which can be applied for microbiology research. AS a distribution company, Amerigo Scientific aims to integrate global superior product resources and technical resources to provide scientists with the latest, professional solutions.

“This new product further strengthens our microbiology research materials offering, and we are ready to meet our customers’ increasing demand for single-use research tools,” said Nina Cooper, Ph.D., the chief scientist at Amerigo Scientific. “We will continue to expand our portfolio to give us the ability to work closely with our customers, and provide more access to customized solutions for them. With our professional team, equipped with perfect technical support and thoughtful customer service, we are always ready to provide individualized customer service with high standards.”

For more information about the HHD BROTH or any other details about Amerigo Scientific’s research products, please visit https://www.amerigoscientific.com.

About Amerigo Scientific

Amerigo Scientific is a distribution company that focuses on providing critical products and services to the biomedical and life science research communities. Although it is a new company, Amerigo Scientific’s founder has had more than 20 years of rich experience in the biomedical and biochemical fields, and has established close contacts with key personnel in top international pharmaceutical and biotech companies, academic research institutes, and government research agencies. It has a professional team, as most of its employees own a graduate (Ph.D. or master) degree in life science, so they can understand customers’ questions or concerns and are always ready to provide individualized customer service with high standard.

Amerigo Scientific Supports Microbiology Research with the Launch of HHD BROTH

STEMart, a provider of integrated medical device CRO services dedicated to medical device and diagnostic clinical development, expands its Biocompatibility Testing services and introduces biocompatibility evaluation of breathing gas pathways in medical devices. This new testing follows the biocompatibility guidelines modified for medical devices and uses a solid scientific rationale.

Biocompatibility evaluation of breathing gas pathways in medical devices is a powerful tool for assessing contaminants from the air emitted from the device for Volatile Organic Compounds (VOCs) and particulate matter when the gas pathway is intended to contact a patient’s respiratory tract. STEMart now offers various services for Testing of Breathing Gas Pathways in medical devices.

For the biocompatibility standard, “Chemical Characterization of medical device materials”, part eighteen of the Biological evaluation of medical device standards(ISO 10993-18), gives the general guidance for E/L testing including polar, semi-polar, and nonpolar solvents measure leaching of organic compounds. It also outlines that VOCs can be determined by analyzing water extracts or directly analyzing test items by heating the headspace.

ISO 18562 is a biocompatibility assessment standard for materials or medical devices with breathing gas pathways. It provides instructions for E/L extraction using purified water to simulate humidification vapor condensate, and VOC analysis for VOCs emitted in a gas stream under simulated use conditions (temperature, flow rate, duration) by GC/MS method, and particulates matter analysis of PM2.5 and PM10 emissions. Notably, the choice of sampling flow rate and temperature should be clinically relevant.

STEMart now offers biocompatibility evaluation of material or medical device with a breathing gas pathway utilized modern analytical techniques and instrumentation, following the biocompatibility guidelines modified for medical devices and using a solid scientific rationale. For example, STEMart can measure particulate matter emitted from medical equipment, flowing clean and dry air over the test material and collecting particulates from the output, and calculating PM2.5 and PM10 particle levels and recording minimum, maximum and average particle concentrations.

As for VOC analysis with Gas Chromatography Mass Spectrometry (GC/MS), STEMart offers two options for collecting and sampling VOCs: thermal desorption tube/can method and stainless steel SUMMA can method. VOCs are collected by directing clean, dry air through the test material in a controlled environment. Subsequently, particulate matter is sampled from the output. VOCs in gas samples ca be analyzed by GC/MS. VOC concentrations are reported in µg/m3. CO, CO2 and ozone can also be analyzed.

If you have additional questions about biocompatibility evaluation of breathing gas pathways in medical devices or if you would like to find out more about the medical device development service, please visit https://www.ste-mart.com.

About STEMart

STEMart is an industry-leading eCommerce platform with an expanded global footprint and has a broad portfolio of more than 10,000 products. It aims to provide best-in-class lab materials, medical instruments and consumables, excellent technologies, and high-quality services to global customers in the fields of science, technology, and engineering, from the discovery stage towards the manufacturing process. STEMart is dedicated to making research and biotech production simpler and safer, and through that to accelerate access to better health for people everywhere.

STEMart Introduces Biocompatibility Evaluation of Breathing Gas Pathways in Medical Devices

Plant-mediated routes for synthesising metal oxide nanoparticles are gaining tremendous attention due to the benefits of the technique: simplicity, cost-effectiveness, and eco-friendliness. We compared the properties of zinc oxide nanoparticles (ZnONPs) made from aqueous leaf extracts of Chromolaena odorata and Manihot esculenta, both of which are abundant on the African continent. The phytochemical composition of the extracts was first assessed using gas chromatography-mass spectrometry (GC-MS) to determine the types of biomolecules involved in the reducing and capping processes that result in ZnONP formation. After that, UV-Vis spectrophotometry, scanning electron microscopy, energy dispersive X-ray analysis, transmission electron microscopy, X-ray diffractometry, and Fourier transform infrared spectroscopy (FTIR) were used to study ZnONP formation, morphological characteristics, elemental composition, shape and size properties, and phase composition. The ZnONPs made with Chromolaena odorata leaf extract had a better distribution of spherical and hexagonal forms, with an average particle size of 42.35 nm. The ZnONPs made with Manihot esculenta leaf as a reductant had a particle size of 14.71 nm on average and were more agglomerated with poor particle distribution. Phytosterols were shown to be the most important biomolecules in the reduction and capping reactions, according to GC-MS and FTIR analyses. In this study, we created a cost-effective technique for the synthesis of eco-friendly ZnONPs for diverse applications, particularly in Africa, using Chromolaena odorata and Manihot esculenta leaves.

Characterisation of ZnO nanoparticles prepared using aqueous leaf extracts of Chromolaena odorata (L.) and Manihot esculenta (Crantz)

**Materials**

The Chromolaena odorata and Manihot esculenta leaves utilised in this study were obtained from a bush near Bells University of Technology in Ota, Ogun State, Nigeria, and their identities were confirmed at the University of Lagos’ Botany Department. Sigma-Aldrich (Gillingham, UK) provided the reagents utilised, which included Zn(NO3)2.6H2O and absolute ethanol.

**Preparation of the aqueous leaf extracts**

Dust and particle contaminants were removed from the leaves of Chromolaena odorata and Manihot esculenta by washing them first with tap water and then with deionised water to prepare the extracts. They were then dried in the sun and ground into fine granules to speed up the extraction process. Each powdered sample (24 g) was combined separately with 250 mL deionised water, sealed, and heated in a water bath for 15 min. The combinations were then cooled to ambient temperature and filtered to yield the aqueous extracts, which were then stored at 5–10 °C in the refrigerator for later use.

**Synthesis of ZnONPs**

The Chromolaena odorata and Manihot esculenta aqueous leaf extracts (250 mL) were added in drops to a 0.1-M solution of Zn(NO3)2.6H2O (50 mL) in a conical flask under a continual stirring condition using a magnetic stirrer to make ZnONPs. The 12-h addition time resulted in the production of solid-liquid dispersion mixtures, which were centrifuged for 15 min at 3835 g force. The supernatants were then removed, and the residues were rinsed with deionised water to remove any remaining Zn(NO3)2.6H2O and organic compounds. For drying, the remnants were placed in a 70 °C oven for 2 h. After that, ZnONPs were obtained by calcination in a muffle furnace at 500 °C for 3 h. To improve the dispersion of the ZnONPs, they were sonicated in ethanol at 40 °C for 1 h in an ultrasonic cleaner. ZnONPs from Chromolaena odorata were designated ZnO_CNPs, while those from Manihot esculenta were designated ZnO_MNPs.

**Characterisation**

The phytochemicals in the Chromolaena odorata and Manihot esculenta aqueous leaf extracts were determined using a gas chromatograph–mass spectrometer (GC-MS; Shimadzu QP2010SE, Markham, Ontario, Canada). Before injection into the GC-MS, ethanol was added to the crude aqueous extracts to obtain an ethanolic solution of the extracts at a mixture ratio of 1:1. From the retention times, fragmentation patterns and peak areas, the various phytochemicals present in the extracts and their concentrations were determined. The formation of ZnONPs was validated using a UV-Vis absorption spectrophotometer to measure the wavelengths of absorption of the mixture obtained during the reaction between Zn(NO3)2.6H2O and the Chromolaena odorata and Manihot esculenta leaf extracts, which ranged from 300 nm to 800 nm (Uniscope SM 7504).

In a scanning electron microscope (SEM) equipped with an energy dispersive X-ray analyser (EDX) unit (JEOL JSM 7660F), the microstructure and elemental composition of the ZnONPs were assessed to evaluate particle distribution and support the presence of ZnO in the samples. An accelerating voltage of 15 kV was used to evaluate the material.

“The samples were analysed using a transmission electron microscope (TEM; JEM-ARM200F-G) running at a 200-kV accelerating voltage to determine the particle size and shape of the ZnONPs. The average particle size of each sample was then calculated using ImageJ software from the TEM micrographs.

To establish the type of phases present and analyse the crystalline nature of the ZnONPs, the diffraction patterns of the samples were obtained using an X-ray diffractometer (XRD; Rigaku D/Max-IIIC).

Fourier transform infrared (FTIR) spectroscopy (Nicolet iS10) with a wavenumber in the range of 350–4000 cm-1 was used to determine the type of bonds present in the ZnONPs to complement the results of the GC-MS, UV-Vis, and XRD.

Preparation of Aqueous leaf extracts, Synthesis of ZnONPs & Characterisation

We analysed four different Sudanese roselle samples for their potential as novel xanthine oxidase (XO) inhibitors. Phytochemical screening showed the presence of polyphenols, flavonoids, organic acids, saponins and sterols in all samples. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to identify and characterise five anthocyanins in all samples: cyanidin-3-glucoside (cy-3-glu), delphinidin-3-sambubioside (dp-3-sam), cyanidin-3-rhamnoside (cy-3-rhm), delphinidin-3-rhamnoside (dp-3-rhm) and pelargonidin-3-glucoside (pg-3-glu). Identification of cy-3-rhm, dp-3-rhm and pg-3-glu confirmed the selectivity and sensitivity of LC-MS as a powerful technique for identifying anthocyanins. In silico studies of the identified anthocyanins were performed to explore their promising inhibitory activity toward XO. Interactions between the ligand and the enzyme were via the H-bond, and hydrophobic (π-alkyl, π-sigma and alkyl) and/or electrostatic (π-cation) bonds. Inhibition of the anthocyanins was compared with that of topiroxostat, a commercial drug for hyperuricaemia. Dp-3-rhm was the most active inhibitor with a binding energy of ca. -10.90 kcal/mol compared to topiroxostat’s binding energy of ca. -8.60 kcal/mol. “The good inhibition results obtained from anthocyanins toward XO suggest their application as a drug candidate to treat gout and other diseases related to the activity of XO.

Phytochemicals and in silico investigations of Sudanese roselle

**_Chemicals_**

All chemicals were of analytical grade except those used in LC-MS analysis which were HPLC-grade.”

_**Phytochemical screening**_

**Preparation of extracts**

The extract was prepared according to Wrolstad et al.’s18 alternative protocol with some modifications in the purification step. The purification was established by simple liquid-liquid extraction using an organic non-polar solvent (petroleum ether) and (0.01% (v/v) HCl) acidified methanol to remove water-insoluble impurities. The desired layer was reserved, the solvent was evaporated using a rotatory evaporator (Heidolph Laborota 4000 efficient HB digital, Germany) and allowed to air dry for 3–4 h. The attained gummy extract was used for all the phytochemical qualitative and quantitative analyses after dissolving in 1–2 cm3 methanol for each qualitative test.

**Preliminary screening**

Qualitative analysis was carried out according to methods described by Harborne10 and Sofowara19 via simple test tube tests. These are the test for polyphenols (FeCl3 test), test for flavonoids (AlCl3 test), test for sterols (H2SO4 test), test for saponins (Frothing test), and test for anthraquinones (KOH test).

**Thin-layer chromatography**

An amount of 10 mg from the gummy powdered extract of each of the samples was dissolved in 1 cm3 of methanol and, using a capillary tube, spots were made from each sample extract on an aluminium sheet of pre-coated silica gel thin-layer chromatography plates, then elution was carried out in a covered thin-layer chromatography chamber using the mobile phase of a mixture of chloroform and methanol (7:3 v/v) according to Harborne10. The procedure was repeated on two other plates.

After performing the elution, plates were examined immediately in daylight, then sprayed with chemical reagents and visualised under a UV lamp (at 365 nm). Bromocresol green was the spray reagent employed for detection of organic acids, while AlCl3 was used for detection of flavonoids.20

**Quantitative analysis**

Total phenolic content was measured using Folin–Ciocalteu’s reagent as described by Wolfe et al.21 Total flavonoid and total anthocyanin contents were measured according to Shanmukha et al.22 and Pacôme et al.6, respectively.

For total phenolic and flavonoid contents, absorbance was measured at 765 nm and 510 nm, respectively, against a reagent blank using a Jenway 7205 UV/Vis double-beam spectrophotometer. The concentrations of phenolics and flavonoids in the test sample were determined and expressed as gram equivalent of gallic acid (g GAE) and quercetin per gram of air-dried extract (g ADE), using a standard calibration plot for each individually. All measures were performed in duplicate.

Determination of total anthocyanins

Total anthocyanin content was measured at an absorbance of 530 nm using a Jenway 7205 UV/Vis spectrophotometer against the blank; total anthocyanin content was estimated as cyanidin-3-glucoside at 530 nm using a molar extinction coefficient of 26 900 L/mol/cm) and molar mass (449 g/mol) and was expressed as cyanidin-3-glucoside (g cy-3-glu)/(100 g of air-dried extract).

**LC-MS/MS identification**

Anthocyanin extraction was carried out using 250 cm3 acidified methanol by Soxhlet apparatus2 at a temperature of 50–55 °C. The extract was purified by simple liquid/liquid extraction after reducing the extract volume using the rotatory evaporator (Heidolph Laborota 4000 efficient HB digital, Germany).

Anthocyanins were identified in the purified sample extracts using LC-MS/MS with a UPLC-Q-TOF-MS (XEVO-G2 QTOF YCA119, Waters, using Acquity UPLC HS T3 (100 × 2.1 mm, 1.8 μm; Waters) column maintained at 45 °C in a column oven in the LC-MS Laboratory, Beijing University of Chemical Technology BUCT, China. The analysis was carried out according to the method described by Cahlíková et al.23 with some modifications. The conditions of the chromatographic run were as follows: 0–1 min: 90% A and 10% B, 1–5 min: 85% A and 15% B, 5–10 min: 40% A and 60% B, 10–13 min: 0% A and 100%B, 13–15 min: 90% A and 10% B. Gradient mobile phase composed of water acidified with 0.1% formic acid (solvent A) and acetonitrile (solvent B). The flow rate and sample injection volume were 0.4 cm3/min and 3.00 µL, respectively.

The ion source of the quadropole time-of-flight (Q-TOF) detector was set at the positive electrospray ionisation polarity mode. The optimum conditions of the Q-TOF system, performed in the resolution mode, were: capillary voltage: +3.000 kV, ion source temperature: 100 °C, extractor: ٤.٠ V; RF lens: ٠.٣ V, mass range m/z: 50 to 2000 Da, nitrogen as the desolvation gas at a flow rate of 800 L/h and at a temperature of 400 °C. Nitrogen was used also as the cone gas (٢٠ L/h), and argon as the collision gas. The cone voltage, collision energy, and well time were carefully optimised for each compound and transition individually (cone voltage set at 4/30 V, collision energy set finally at 6 eV). The software used for the MS control and data gathering was Mass Lynx 4.1.

**Molecular docking**

Molecular docking is a method used to screen for the ability of specific small compounds (ligands) to fit into a previously identified therapeutic target (the receptor; usually a protein enzyme).24,25 Several docking programs can be used: AutoDock Vina, GOLD, and MOE-Dock are top ranked with the best scores.25 We used the AutoDock Vina program26 for the docking of anthocyanin ligands to the active site of the XO enzyme, after performing the required preparations.

First, the protein crystal structure and ligand 3D structures were downloaded from RCSB Protein Data Bank27 (1FIQ PDB code) and PubChem online databases28, respectively. Then the suitable binding site of the protein was obtained using Chimera software29 and the ligands were converted via Open Babel software30 from SDF format to pdbqt. Thereafter, using autodock tools31, all water molecules in the protein pdb file were removed, hydrogen atoms were added, and the grid box dimensions were set, then the file saved as pdbqt. Ligands were prepared by detecting torsions for each structure and saved as a pdbqt file. The AutoDock Vina order was then created after preparing the configuration files. The process was repeated for each docking trial for all ligands.

**Data analysis**

The obtained (out.pdbqt) file for each ligand was visualised using discovery studio32 and pymol33 visualisation programs.

Chemicals & Phytochemical Screening (Thin-layer Chromatography, Molecular Docking, LC-MS/MS identification)

 Circularize DNA oligos of very small size - 

The g-complex loads the b-clamp to the p/t and b-clamp then slides on the DNA bidirectionally. In order to ensure that the b-clamp does not slide off the edge of the p/t one can either block the ends of the DNA using biotin-streptavidin or use circular DNA. In our unpublished experiments, we see significant inhibition of polymerase activity due to the presence of bulky groups like streptavidin, hence we proceeded to use circular template for our work. 

Observing in-vitro DNA synthesis using beta clamp

DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA) and their sequences. All oligomers were purified in-house using denaturing PAGE purification. The templates contained a 5’ phosphate modification in order to circularize the template. Briefly, the template to be circularized was annealed to a 20 mer linker, which was complementary to the first ten and the last ten nucleotides of the linear template. After annealing, we used T4 DNA ligase on the template/linker to connect the 5’-P and 3’-OH, forming a circle. The circle was then purified of its linear product and the linker by running through a PAGE gel and subsequent extraction.

- circular template - 5' Phosphate modification
- linker DNA 2X Circular template :**  **complementary to the first ten and the last ten nucleotides of the linear template.
- Anneal
- Add T4 ligase 1 microL for 1 microgram
- 20Hours 20C
- ethanol precipitation
- **purify on 10% PAGE gel - poured into a SDS protein gel**
-  

small DNA/oligos Circularization and purification

Mass spectrometry is a commonly identification method for the products of a protein digest, modification, purification, or the like. Sample preparation is handled via Ziptips, an extremely fast (if not expensive) method of reducing contaminants in your sample.

ZipTipping solutions

A. 	0.2% TFA in HPLC grade water; for dilution of protein digests.

B.	0.1% TFA in 50% ACN/50% water; for peptide elution.

C. 	100% acetonitrile (ACN); wetting agent for ZipTip.

D. 	0.1% TFA in water; for equilibration of ZipTip

 

1. For each unknown, label a 0.65 ml tube as a diluted sample. Combine 6.0 µl of solution A and 4.0 µl of your protein sample. Gently mix, and then cap the tube. 

2. Transfer 2.5 µl of solution B to the bottom of the tubes and cap them. Label as elution tubes

3. Attach a ZipTip to your pipettor and set volume to 8 µl.  Draw up 8 µl of solution C and dispense onto a Kimwipe.  Keep the plunger down, and repeat once.  Proceed to step #4 without letting the plunger retract, i.e. without sucking air through the C18 material.

4. With the plunger still down, draw up 10 µL of solution D and dispense onto a Kimwipe. Keep the plunger down, and repeat 2X.  Proceed to step #5 without letting the plunger retract.

5. Slowly and carefully cycle 8 µl of your diluted sample from #1 above through the equilibrated ZipTip.  Do 10 complete cycles while keeping the tip immersed to bind the proteins to the tip. At the end of your last cycle, dispense the liquid into a Kimwipe. 

6. Place the tip in solution D and draw up 8 µl.  Dispense onto a Kimwipe and repeat 3X.  This step is critical for removing non-peptide components. After the last wash, make sure all the liquid is out of the tip.

7. Set the pipette for 2.0 µl and elute the peptides by cycling the solution B in the PET tube (from step 2) through the tip for a total of 6 cycles.  Cap the tube tightly and store at -15 to -20°C.

MALDI-TOF Protein Prep

A common protocol for the purification of polyhistidine tagged proteins uses an Immobilized metal affinity column (IMAC). This protocol for affinity purification is a generalization that utilizes Amersham sepharose beads and nickel ions.

Buffers
Binding Buffer -  pH 8.0 with monosodium phosphate and NaOH, and 20mM Imidazole to reduce nonspecific binding. 

Wash Buffer - pH 8.0 with monosodium phosphate and NaOH, and 60mM Imidazole to reduce nonspecific binding.  

Elution Buffer - pH 8.0 with monosodium phosphate and NaOH, and 400mM Imidazole to reduce nonspecific binding. You can optionally use a pH 6.0 buffer that elutes the metal ions from the agarose beads, but this introduces metal ion contamination to the elution.

Column Preparation
1. In 4 C cold room, set up the column on a vertical support. 

2. Flush the column with cold ddH2O. Leave some water in the column. 

3. Resuspend the Amersham sepharose. Transfer the sepharose to the column using a pipette with the tip cut off. 

4. Open the outlet for the column to allow the sepharose to pack at the bottom. Do not let the sepharose dry out.

5. Wash the column with 5 volumes of ddH2O. Drain the last wash to just above the sepharose.

6. Add two column volumes of 0.1M NiSO4. Drain the NiSO4.

7. Wash the column with 5 column volumes of ddH2O until the sepharose is blue-green.

8. Wash the column with 5 column volumes of binding buffer until the sepharose is blue. 

Lysate Preparation

1. Thaw cell pellet from 250 ml culture.

2. Resuspend pellet in 15 ml Binding buffer and 1.0 mM PMSF (150 ul of 100mM).

    a. Binding buffer is pH 8.0 with monosodium phosphate and NaOH, and 20mM Imidazole to reduce nonspecific binding. 

4. Add the resuspension to prechilled oakridge tubes. 

5. Sonicate the cell suspension 10 seconds at a time, with 10 seconds in between sonications. Repeat 7 times.

6. Centrifuge the lysate in SS34 rotor at 18,000 rpm (39,000 RPM) for 20 minutes at 4 oC.

7. Filter the lysate supernatant through a 0.22 um syringe filter.

Immobilized Metal Ion Affinity Chromatography

1. Apply lysate to the prepared column. Collect the flow through in a tube and reapply to the column.

2. Repeat step 1 two more times. 

     a. Optionally, you can save 100 ul of the flow through for analyzing initial binding conditions. If so, add 2X laemmli buffer to the 100 ul and freeze at -20 oC.

3. Prepare 50 ml (10 column volumes) of wash buffer with 0.1 mM PMSF (500 ul).

4. Add the wash buffer to the column, draining 5 ml fractions.

     a. Optionally save these fractions by freezing them at -80 oC.

5. For fractions 2, 5, and 8, transfer 10 ul of the fraction to a microfuge tube and add 2X Laemmli buffer. Freeze these tubes at - 20 oC.

6. Prepare 10 ml elution buffer (10 column volumes) with 0.1mM PMSF (250 ul).

7. Add 5 ml elution buffer to the column and allow it to incubate for 5 minutes before allowing it to wash through. 

8. Collect the elution in 1 ml fractions using 13x100 mm disposable glass tubes. Save all fractions by freezing them at -80 oC.

     a. Optionally save 10 ul of each elution fraction by adding 10 ul 2X Laemmli buffer in a microfuge tube. Freeze these tubes at -20 oC.

Wash the column with 10 volumes of binding buffer. If storing the column for a long period of time, wash the column with ddH2O and store the sepharose in 20% ethanol.

IMAC Protein Purification

Here is a scraping protocol for large scale induction and harvesting of transformed E. coli (typically BL21 or NiCo21 strains), starting with already transformed colonies. For expression of some proteins, scraping bacterial lawns from plates seems to increase the overall yield. Frozen cell pellets can be stored almost indefinitely in a -80 freezer until they are needed for lysis and protein purification. 

1. Inoculate 3 transformed colonies in 200 ul LB-amp-cam broth. 

2. Plate the 200 ul broth mixture on LB-amp-cam plates. Incubate overnight at 37 oC. 

3. You can prepare additional plates in this step if desired to decrease the time needed to reach the necessary optical density

4. Prewarm 1 L LB amp/cam in a 2 L fernbach flask at 37 oC. 

5. Scrape culture from plate media to the broth until an OD600 of 0.1 is reached. .

6. Grow the cells at 37 oC on a 225 RPM shaker to an OD600 of 0.5-0.8. Check the OD600 frequently using 1 ml of culture in a cuvette to prevent overgrowth.

7. Add 10 ml 100 mM IPTG to the 1 L culture and grow for 2 hours.

8. Spin cells down 200 ml at a time in 250 ml sorvall bottles using the Sorvall RC5 centrifuge. Spin for 5 minutes at 6500 G and 4 oC.  Perform multiple spins to spin down all culture media.

9. Freeze cell pellets at -80 C.

IPTG Induction for Protein Expression

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