ethylenediaminetetraacetic acid  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher ethylenediaminetetraacetic acid
    Ethylenediaminetetraacetic Acid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethylenediaminetetraacetic acid/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ethylenediaminetetraacetic acid - by Bioz Stars, 2020-03
    99/100 stars

    Images

    Related Articles

    Centrifugation:

    Article Title: Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes
    Article Snippet: Frozen adipose tissue biopsies (~100 mg) were homogenized in a buffer containing 50 mM HEPES, 20 mM NaF, 2 mM NaOV, 5 mM EDTA, 5 mM N -acetylmethionine, 10 µM trichostatin A, protease inhibitor cocktail (HALT; Thermo Specific, Waltham, MA), and 5% SDS in a Precellys 24 homogenizer (Bertin Technologies, Montigny-Le-Bretonneux, France) for 2 × 30 s at 6,000 revolutions/min (rpm). .. Samples were subsequently thermomixed (Eppendorf, Hamburg, Germany) at 37°C and 900 rpm for 1 h. The lipid fraction was isolated by centrifugation at 14,000 g for 20 min at room temperature, and the infranatant was collected and stored at −80°C until used for Western blot analysis.

    Article Title: Mapping accessible chromatin regions using Sono-Seq
    Article Snippet: .. HeLa S3 cells were collected by centrifugation, resuspended in digestion buffer [100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0) and 0.5% SDS] and digested overnight at 50 °C with 0.1 mg/mL proteinase K (Ambion). .. The DNA was recovered, treated with RNase (Qiagen) for 3 h at 37 °C, extracted once with phenol-chloroform, once with chloroform, ethanol precipitated and resuspended at 2.5 × 108 cell equivalents in 5 mL of 1X Tris-EDTA (TE) pH 7.5 (10 mM Tris, 1 mM EDTA).

    Cytometry:

    Article Title: Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques
    Article Snippet: Paragraph title: Flow cytometry. ... Subsequently, the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm kit (Thermo Fisher Scientific).

    Blocking Assay:

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100. .. Proteins in the supernatant were separated by electrophoresis on 7.5% SDS-PAGE and then transferred to nitrocellulose membrane that was kept overnight at 4°C in blocking buffer (AquaBlock, East Coast Biologics, North Berwick, ME).

    SYBR Green Assay:

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion). .. The immunoprecipitated DNA and the input were used as templates for quantitative PCR (qPCR) reactions conducted with ABI 7500 instrumentation (Applied Biosystems, Foster City, CA) and SYBR Green PCR Master Mix (Life Technologies).

    Incubation:

    Article Title: Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes
    Article Snippet: Frozen adipose tissue biopsies (~100 mg) were homogenized in a buffer containing 50 mM HEPES, 20 mM NaF, 2 mM NaOV, 5 mM EDTA, 5 mM N -acetylmethionine, 10 µM trichostatin A, protease inhibitor cocktail (HALT; Thermo Specific, Waltham, MA), and 5% SDS in a Precellys 24 homogenizer (Bertin Technologies, Montigny-Le-Bretonneux, France) for 2 × 30 s at 6,000 revolutions/min (rpm). .. Protein aliquots were separated by gel electrophoresis using StainFree 4–15% CriterionXT gels (Bio-Rad), electrotransferred to a polyvinylidene fluoride membrane, blocked for 2 h in Tris-buffered saline-Tween (TBS-T) containing 1% bovine serum albumin, and incubated in primary antibody for one to three nights.

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: Protein and cross-linked DNA were eluted in 100 μl of elution buffer (1% SDS and 0.1 M NaHCO3 ) at 65° for 10 min. To reverse the cross-linking, the mixture was incubated at 65° overnight with 50 μg/ml proteinase K. DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 and precipitated with 5 M NaCl, glycogen, and ethanol. .. The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion).

    Article Title: P-glycoprotein Targeted Photodynamic Therapy of Chemoresistant Tumors using Recombinant Fab Fragment Conjugates
    Article Snippet: Pgp-targeting Fab fragment was reacted with IR700-maleimide at molar ratio of 1:2 in phosphate buffer (pH 7.0) containing 1 mM EDTA for 2 h. The resulting conjugates were purified using Zeba™ spin desalting column (40K MWCO, Thermo Fisher Scientific). .. Briefly, Pab was incubated with IR700-NHS at molar ratio of 1:4 in phosphate buffer (pH 8.0) for 1 h. The product of the conjugation was purified using a Zeba™ spin desalting column (40K MWCO).

    Proliferation Assay:

    Article Title: Ovarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic Response to Paclitaxel
    Article Snippet: Paragraph title: Proliferation Assay: ... Cells were trypsinized (0.25% trypsin, EDTA) and counted by a Countess™ Automated Cell Counter (Invitrogen) after 24, 48, and 72 hours in culture.

    BIA-KA:

    Article Title: The glycoprotein GPNMB is selectively elevated in the substantia nigra of Parkinson’s disease patients and increases after lysosomal stress
    Article Snippet: Tissue was mechanically homogenized for 10 seconds in 100µL 10mM Tris buffer (KD-Medical, RGE-3340, pH 7.4) prepared in EmbryoMax Ultrapure water, supplemented with protease inhibitors and 0.5mM EDTA (Thermo Fischer, 78430). .. Total protein concentration in each supernatant sample was determined using the BCA protein quantification kit (Pierce, BCA protein assay kit, 23225).

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100. .. Protein concentration in the supernatant was measured using a Micro BCA Assay Kit (Thermo Scientific Pierce).

    Article Title: P-glycoprotein Targeted Photodynamic Therapy of Chemoresistant Tumors using Recombinant Fab Fragment Conjugates
    Article Snippet: Pgp-targeting Fab fragment was reacted with IR700-maleimide at molar ratio of 1:2 in phosphate buffer (pH 7.0) containing 1 mM EDTA for 2 h. The resulting conjugates were purified using Zeba™ spin desalting column (40K MWCO, Thermo Fisher Scientific). .. The protein concentration of the antibody conjugates were determined with BCA protein assay kit (Thermo Fisher Scientific), and the IR700 concentration was quantified by measurement of the absorption at 689 nm with the spectroscopy in order to estimate the number of IR700 molecules conjugated to each antibody molecule.

    Article Title: Tuning mitochondrial structure and function to criticality by fluctuation-driven mechanotransduction
    Article Snippet: .. Measurement of mitochondrial proteins Total proteins were extracted with cell lysis buffer (Sigma-Aldrich Inc.) in the presence of protease inhibitors (Halt™ Protease Inhibitor Cocktail (100×) and EDTA (100×), ThermoFisher Scientific) and quantified according to the BCA method (Pierce). .. Equal amounts (~3.8 µg) of protein/sample were separated on 4–20% gradient SDS-polyacrylamide gels (Bio-Rad Laboratories).

    Western Blot:

    Article Title: Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes
    Article Snippet: Western Blot analyses were used to assess total and phosphorylated (p) levels of relevant proteins in adipose tissue biopsies. .. Frozen adipose tissue biopsies (~100 mg) were homogenized in a buffer containing 50 mM HEPES, 20 mM NaF, 2 mM NaOV, 5 mM EDTA, 5 mM N -acetylmethionine, 10 µM trichostatin A, protease inhibitor cocktail (HALT; Thermo Specific, Waltham, MA), and 5% SDS in a Precellys 24 homogenizer (Bertin Technologies, Montigny-Le-Bretonneux, France) for 2 × 30 s at 6,000 revolutions/min (rpm).

    Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus
    Article Snippet: .. Cell lysates for western blot analysis were produced using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl [pH 7.5], 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with 1% NP-40 detergent (Thermo Fisher) and protease inhibitor (Sigma-Aldrich, Allentown, PA, USA). ..

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: Paragraph title: Western blottng. ... The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100.

    Article Title: Tuning mitochondrial structure and function to criticality by fluctuation-driven mechanotransduction
    Article Snippet: Measurement of mitochondrial proteins Total proteins were extracted with cell lysis buffer (Sigma-Aldrich Inc.) in the presence of protease inhibitors (Halt™ Protease Inhibitor Cocktail (100×) and EDTA (100×), ThermoFisher Scientific) and quantified according to the BCA method (Pierce). .. Western blots were performed with mouse or rabbit antibodies for mitofusin-1 (1/2000, Abcam), mitofusin-2 (0.5 µg/ml, Abcam), DRP1 (1:500, Novus Biologicals), OPA1 (1:1000, Novus Biologicals), ATPB for the β subunit of the ATP synthase (0.5 µg/ml, Abcam) and VDAC1 (1 µg/ml, Abcam).

    Real-time Polymerase Chain Reaction:

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion). .. The immunoprecipitated DNA and the input were used as templates for quantitative PCR (qPCR) reactions conducted with ABI 7500 instrumentation (Applied Biosystems, Foster City, CA) and SYBR Green PCR Master Mix (Life Technologies).

    Conjugation Assay:

    Article Title: P-glycoprotein Targeted Photodynamic Therapy of Chemoresistant Tumors using Recombinant Fab Fragment Conjugates
    Article Snippet: Pgp-targeting Fab fragment was reacted with IR700-maleimide at molar ratio of 1:2 in phosphate buffer (pH 7.0) containing 1 mM EDTA for 2 h. The resulting conjugates were purified using Zeba™ spin desalting column (40K MWCO, Thermo Fisher Scientific). .. Briefly, Pab was incubated with IR700-NHS at molar ratio of 1:4 in phosphate buffer (pH 8.0) for 1 h. The product of the conjugation was purified using a Zeba™ spin desalting column (40K MWCO).

    Flow Cytometry:

    Article Title: Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques
    Article Snippet: Paragraph title: Flow cytometry. ... Subsequently, the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm kit (Thermo Fisher Scientific).

    Immunoprecipitation:

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: Tagged proteins were immunoprecipitated with 5 μg mouse anti-Flag (Sigma) or anti-cMyc (Millipore, Bedford, MA) bound to Dynabeads Protein G for immunoprecipitation (Invitrogen). .. The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion).

    Protease Inhibitor:

    Article Title: Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes
    Article Snippet: .. Frozen adipose tissue biopsies (~100 mg) were homogenized in a buffer containing 50 mM HEPES, 20 mM NaF, 2 mM NaOV, 5 mM EDTA, 5 mM N -acetylmethionine, 10 µM trichostatin A, protease inhibitor cocktail (HALT; Thermo Specific, Waltham, MA), and 5% SDS in a Precellys 24 homogenizer (Bertin Technologies, Montigny-Le-Bretonneux, France) for 2 × 30 s at 6,000 revolutions/min (rpm). .. Samples were subsequently thermomixed (Eppendorf, Hamburg, Germany) at 37°C and 900 rpm for 1 h. The lipid fraction was isolated by centrifugation at 14,000 g for 20 min at room temperature, and the infranatant was collected and stored at −80°C until used for Western blot analysis.

    Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus
    Article Snippet: .. Cell lysates for western blot analysis were produced using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl [pH 7.5], 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with 1% NP-40 detergent (Thermo Fisher) and protease inhibitor (Sigma-Aldrich, Allentown, PA, USA). ..

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: .. The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100. .. The sample was sonicated using a Misonix S3000 (Misonix, Farmingdale, NY) for 1 min (4 strokes of 15 s at 5-s interval) then centrifuged for 30 min at 13,000 g to remove nuclei and large debris.

    Article Title: Tuning mitochondrial structure and function to criticality by fluctuation-driven mechanotransduction
    Article Snippet: .. Measurement of mitochondrial proteins Total proteins were extracted with cell lysis buffer (Sigma-Aldrich Inc.) in the presence of protease inhibitors (Halt™ Protease Inhibitor Cocktail (100×) and EDTA (100×), ThermoFisher Scientific) and quantified according to the BCA method (Pierce). .. Equal amounts (~3.8 µg) of protein/sample were separated on 4–20% gradient SDS-polyacrylamide gels (Bio-Rad Laboratories).

    Dissection:

    Article Title: The glycoprotein GPNMB is selectively elevated in the substantia nigra of Parkinson’s disease patients and increases after lysosomal stress
    Article Snippet: Mice were euthanized with sodium pentobarbital and subsequently transcardially perfused with 0.9% heparinized saline (for fresh dissection) or followed by 4% PFA (for fixed brain harvest) 24 hours after the final administration of DMSO or CBE. .. Tissue was mechanically homogenized for 10 seconds in 100µL 10mM Tris buffer (KD-Medical, RGE-3340, pH 7.4) prepared in EmbryoMax Ultrapure water, supplemented with protease inhibitors and 0.5mM EDTA (Thermo Fischer, 78430).

    Inhibition:

    Article Title: The glycoprotein GPNMB is selectively elevated in the substantia nigra of Parkinson’s disease patients and increases after lysosomal stress
    Article Snippet: Paragraph title: Pharmacological inhibition of lysosomal glucocerebrosidase ... Tissue was mechanically homogenized for 10 seconds in 100µL 10mM Tris buffer (KD-Medical, RGE-3340, pH 7.4) prepared in EmbryoMax Ultrapure water, supplemented with protease inhibitors and 0.5mM EDTA (Thermo Fischer, 78430).

    Protein Concentration:

    Article Title: The glycoprotein GPNMB is selectively elevated in the substantia nigra of Parkinson’s disease patients and increases after lysosomal stress
    Article Snippet: Tissue was mechanically homogenized for 10 seconds in 100µL 10mM Tris buffer (KD-Medical, RGE-3340, pH 7.4) prepared in EmbryoMax Ultrapure water, supplemented with protease inhibitors and 0.5mM EDTA (Thermo Fischer, 78430). .. Total protein concentration in each supernatant sample was determined using the BCA protein quantification kit (Pierce, BCA protein assay kit, 23225).

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100. .. Protein concentration in the supernatant was measured using a Micro BCA Assay Kit (Thermo Scientific Pierce).

    Article Title: P-glycoprotein Targeted Photodynamic Therapy of Chemoresistant Tumors using Recombinant Fab Fragment Conjugates
    Article Snippet: Pgp-targeting Fab fragment was reacted with IR700-maleimide at molar ratio of 1:2 in phosphate buffer (pH 7.0) containing 1 mM EDTA for 2 h. The resulting conjugates were purified using Zeba™ spin desalting column (40K MWCO, Thermo Fisher Scientific). .. The protein concentration of the antibody conjugates were determined with BCA protein assay kit (Thermo Fisher Scientific), and the IR700 concentration was quantified by measurement of the absorption at 689 nm with the spectroscopy in order to estimate the number of IR700 molecules conjugated to each antibody molecule.

    Polymerase Chain Reaction:

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion). .. The immunoprecipitated DNA and the input were used as templates for quantitative PCR (qPCR) reactions conducted with ABI 7500 instrumentation (Applied Biosystems, Foster City, CA) and SYBR Green PCR Master Mix (Life Technologies).

    Sonication:

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100. .. The sample was sonicated using a Misonix S3000 (Misonix, Farmingdale, NY) for 1 min (4 strokes of 15 s at 5-s interval) then centrifuged for 30 min at 13,000 g to remove nuclei and large debris.

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: The chromatin in the lysates was sheared by sonication with 30 cycles (effective sonication time: 3 min 45 sec) at 20% amplitude in an Episonic multi-functional bioprocessor Model Oasis 180. .. The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion).

    Article Title: Mapping accessible chromatin regions using Sono-Seq
    Article Snippet: HeLa S3 cells were collected by centrifugation, resuspended in digestion buffer [100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0) and 0.5% SDS] and digested overnight at 50 °C with 0.1 mg/mL proteinase K (Ambion). .. HeLa S3 cells were collected by centrifugation, resuspended in digestion buffer [100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0) and 0.5% SDS] and digested overnight at 50 °C with 0.1 mg/mL proteinase K (Ambion).

    Injection:

    Article Title: The glycoprotein GPNMB is selectively elevated in the substantia nigra of Parkinson’s disease patients and increases after lysosomal stress
    Article Snippet: Animals were weighed daily to determine appropriate injection volume, and to monitor animals for treatment-induced weight loss. .. Tissue was mechanically homogenized for 10 seconds in 100µL 10mM Tris buffer (KD-Medical, RGE-3340, pH 7.4) prepared in EmbryoMax Ultrapure water, supplemented with protease inhibitors and 0.5mM EDTA (Thermo Fischer, 78430).

    Recombinant:

    Article Title: Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques
    Article Snippet: For Tfh cell analyses, cells were stimulated with recombinant gp140. .. Subsequently, the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm kit (Thermo Fisher Scientific).

    DC Protein Assay:

    Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus
    Article Snippet: Cell lysates for western blot analysis were produced using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl [pH 7.5], 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with 1% NP-40 detergent (Thermo Fisher) and protease inhibitor (Sigma-Aldrich, Allentown, PA, USA). .. Cell lysates for western blot analysis were produced using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl [pH 7.5], 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with 1% NP-40 detergent (Thermo Fisher) and protease inhibitor (Sigma-Aldrich, Allentown, PA, USA).

    Nucleic Acid Electrophoresis:

    Article Title: Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes
    Article Snippet: Frozen adipose tissue biopsies (~100 mg) were homogenized in a buffer containing 50 mM HEPES, 20 mM NaF, 2 mM NaOV, 5 mM EDTA, 5 mM N -acetylmethionine, 10 µM trichostatin A, protease inhibitor cocktail (HALT; Thermo Specific, Waltham, MA), and 5% SDS in a Precellys 24 homogenizer (Bertin Technologies, Montigny-Le-Bretonneux, France) for 2 × 30 s at 6,000 revolutions/min (rpm). .. Protein aliquots were separated by gel electrophoresis using StainFree 4–15% CriterionXT gels (Bio-Rad), electrotransferred to a polyvinylidene fluoride membrane, blocked for 2 h in Tris-buffered saline-Tween (TBS-T) containing 1% bovine serum albumin, and incubated in primary antibody for one to three nights.

    Fluorescence:

    Article Title: Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques
    Article Snippet: Subsequently, the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm kit (Thermo Fisher Scientific). .. Both compensation controls (OneComp eBeads; Thermo Fisher Scientific) and fluorescence minus one (FMO) controls were utilized.

    Isolation:

    Article Title: Growth hormone acts along the PPARγ-FSP27 axis to stimulate lipolysis in human adipocytes
    Article Snippet: Frozen adipose tissue biopsies (~100 mg) were homogenized in a buffer containing 50 mM HEPES, 20 mM NaF, 2 mM NaOV, 5 mM EDTA, 5 mM N -acetylmethionine, 10 µM trichostatin A, protease inhibitor cocktail (HALT; Thermo Specific, Waltham, MA), and 5% SDS in a Precellys 24 homogenizer (Bertin Technologies, Montigny-Le-Bretonneux, France) for 2 × 30 s at 6,000 revolutions/min (rpm). .. Samples were subsequently thermomixed (Eppendorf, Hamburg, Germany) at 37°C and 900 rpm for 1 h. The lipid fraction was isolated by centrifugation at 14,000 g for 20 min at room temperature, and the infranatant was collected and stored at −80°C until used for Western blot analysis.

    Size-exclusion Chromatography:

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: The chromatin in the lysates was sheared by sonication with 30 cycles (effective sonication time: 3 min 45 sec) at 20% amplitude in an Episonic multi-functional bioprocessor Model Oasis 180. .. The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion).

    Labeling:

    Article Title: Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques
    Article Snippet: The panels included the following fluorochrome-labeled antibodies: CD8 (Qdot655), α4β7 (phycoerythrin [PE]), and CXCR5 (PE), all obtained from the Nonhuman Primate Reagent Resource; CD69 (BV737, clone FN50) and FoxP3 (PECy5, clone PCH101), obtained from eBioscience; IL-21 (BV421, clone 3A3-N2.1), CD45 (BV786, D058-1283), and CD3 (clone SP34-2, PE-Cy7 labeled), all from BD Bioscience (San Jose, CA); and CD4 (Pacific Blue, clone OKT4), from Thermo Fisher Scientific (Waltham, MA). .. Subsequently, the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm kit (Thermo Fisher Scientific).

    Mouse Assay:

    Article Title: The glycoprotein GPNMB is selectively elevated in the substantia nigra of Parkinson’s disease patients and increases after lysosomal stress
    Article Snippet: Mice were euthanized with sodium pentobarbital and subsequently transcardially perfused with 0.9% heparinized saline (for fresh dissection) or followed by 4% PFA (for fixed brain harvest) 24 hours after the final administration of DMSO or CBE. .. Tissue was mechanically homogenized for 10 seconds in 100µL 10mM Tris buffer (KD-Medical, RGE-3340, pH 7.4) prepared in EmbryoMax Ultrapure water, supplemented with protease inhibitors and 0.5mM EDTA (Thermo Fischer, 78430).

    Sequencing:

    Article Title: Mapping accessible chromatin regions using Sono-Seq
    Article Snippet: Paragraph title: Preparation of Naked DNA for Sequencing. ... HeLa S3 cells were collected by centrifugation, resuspended in digestion buffer [100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0) and 0.5% SDS] and digested overnight at 50 °C with 0.1 mg/mL proteinase K (Ambion).

    Spectroscopy:

    Article Title: P-glycoprotein Targeted Photodynamic Therapy of Chemoresistant Tumors using Recombinant Fab Fragment Conjugates
    Article Snippet: Pgp-targeting Fab fragment was reacted with IR700-maleimide at molar ratio of 1:2 in phosphate buffer (pH 7.0) containing 1 mM EDTA for 2 h. The resulting conjugates were purified using Zeba™ spin desalting column (40K MWCO, Thermo Fisher Scientific). .. The protein concentration of the antibody conjugates were determined with BCA protein assay kit (Thermo Fisher Scientific), and the IR700 concentration was quantified by measurement of the absorption at 689 nm with the spectroscopy in order to estimate the number of IR700 molecules conjugated to each antibody molecule.

    Lysis:

    Article Title: Computational and Biochemical Studies of Isothiocyanates as Inhibitors of Proteasomal Cysteine Deubiquitinases in Human Cancer Cells
    Article Snippet: .. The lysis buffer was prepared using 1 mM Tris-HCL (pH 8.0), 1M NaCl, 10% NP-40, 0.5M EDTA in distilled water, and Halt™ protease and phosphatase inhibitor cocktail (Thermo Scientific) was added to the whole lysis buffer in 1:100 ratio. ..

    Article Title: Tuning mitochondrial structure and function to criticality by fluctuation-driven mechanotransduction
    Article Snippet: .. Measurement of mitochondrial proteins Total proteins were extracted with cell lysis buffer (Sigma-Aldrich Inc.) in the presence of protease inhibitors (Halt™ Protease Inhibitor Cocktail (100×) and EDTA (100×), ThermoFisher Scientific) and quantified according to the BCA method (Pierce). .. Equal amounts (~3.8 µg) of protein/sample were separated on 4–20% gradient SDS-polyacrylamide gels (Bio-Rad Laboratories).

    Article Title: Autophagy Ablation in Adipocytes Induces Insulin Resistance and Reveals Roles for Lipid Peroxide and Nrf2 Signaling in Adipose-Liver Crosstalk
    Article Snippet: .. All other mouse tissues were solubilized in RIPA lysis buffer containing10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 10% glycerol, 1% NP-40, 0.5% Triton X-100 and protease inhibitors. .. Protein concentration was measured using a BCA protein assay kit (Pierce).

    Purification:

    Article Title: P-glycoprotein Targeted Photodynamic Therapy of Chemoresistant Tumors using Recombinant Fab Fragment Conjugates
    Article Snippet: .. Pgp-targeting Fab fragment was reacted with IR700-maleimide at molar ratio of 1:2 in phosphate buffer (pH 7.0) containing 1 mM EDTA for 2 h. The resulting conjugates were purified using Zeba™ spin desalting column (40K MWCO, Thermo Fisher Scientific). ..

    Chromatin Immunoprecipitation:

    Article Title: Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata
    Article Snippet: Paragraph title: ChIP assay ... The immunoprecipitates were resuspended in 30 µl of TE [10 mM Tris-Cl (pH 8) and 1 mM EDTA) containing 2 μg/ml RNase cocktail (Ambion).

    SDS Page:

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100. .. Proteins in the supernatant were separated by electrophoresis on 7.5% SDS-PAGE and then transferred to nitrocellulose membrane that was kept overnight at 4°C in blocking buffer (AquaBlock, East Coast Biologics, North Berwick, ME).

    Software:

    Article Title: Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques
    Article Snippet: Subsequently, the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm kit (Thermo Fisher Scientific). .. All the samples were collected on an LSR Fortessa X-20 analyzer (BD Biosciences, San Jose, CA) and were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR).

    Electrophoresis:

    Article Title: Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens
    Article Snippet: The capsule-epithelium was removed from the lens and homogenized in 400 µl of ice-cold RIPA buffer (pH 7.5) that contained (in mM) 50 HEPES, 150 NaCl, 1 EDTA, 10 sodium fluoride, 10 sodium pyrophosphate, 2 sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) and phosphatase inhibitor cocktails 1 and 2 (EMD Millipore, Burlington, MA) diluted 1:100. .. Proteins in the supernatant were separated by electrophoresis on 7.5% SDS-PAGE and then transferred to nitrocellulose membrane that was kept overnight at 4°C in blocking buffer (AquaBlock, East Coast Biologics, North Berwick, ME).

    Agarose Gel Electrophoresis:

    Article Title: Mapping accessible chromatin regions using Sono-Seq
    Article Snippet: HeLa S3 cells were collected by centrifugation, resuspended in digestion buffer [100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0) and 0.5% SDS] and digested overnight at 50 °C with 0.1 mg/mL proteinase K (Ambion). .. HeLa S3 cells were collected by centrifugation, resuspended in digestion buffer [100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0) and 0.5% SDS] and digested overnight at 50 °C with 0.1 mg/mL proteinase K (Ambion).

    Radio Immunoprecipitation:

    Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus
    Article Snippet: .. Cell lysates for western blot analysis were produced using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl [pH 7.5], 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with 1% NP-40 detergent (Thermo Fisher) and protease inhibitor (Sigma-Aldrich, Allentown, PA, USA). ..

    Produced:

    Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus
    Article Snippet: .. Cell lysates for western blot analysis were produced using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl [pH 7.5], 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with 1% NP-40 detergent (Thermo Fisher) and protease inhibitor (Sigma-Aldrich, Allentown, PA, USA). ..

    Concentration Assay:

    Article Title: P-glycoprotein Targeted Photodynamic Therapy of Chemoresistant Tumors using Recombinant Fab Fragment Conjugates
    Article Snippet: Pgp-targeting Fab fragment was reacted with IR700-maleimide at molar ratio of 1:2 in phosphate buffer (pH 7.0) containing 1 mM EDTA for 2 h. The resulting conjugates were purified using Zeba™ spin desalting column (40K MWCO, Thermo Fisher Scientific). .. The protein concentration of the antibody conjugates were determined with BCA protein assay kit (Thermo Fisher Scientific), and the IR700 concentration was quantified by measurement of the absorption at 689 nm with the spectroscopy in order to estimate the number of IR700 molecules conjugated to each antibody molecule.

    Staining:

    Article Title: Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques
    Article Snippet: Dead cells were excluded by using the LIVE-DEAD fixable dead cell stain kit, obtained from Invitrogen (Carlsbad, CA). .. Subsequently, the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm kit (Thermo Fisher Scientific).

    other:

    Article Title: Xylazine-induced reduction of tissue sensitivity to insulin leads to acute hyperglycemia in diabetic and normoglycemic monkeys
    Article Snippet: Samples were collected into an EDTA-washed (0.5 M EDTA, pH 8.0, Gibco, Invitrogen Corporation, Grand Island, NY, USA) 2.5-mL disposable syringe and transferred immediately into a 5-ml Monoject™ blood collection tube containing 7.5 mg EDTA (Sherwood Medical, St. Louis, MO, USA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Thermo Fisher mild immunoprecipitation buffer
    Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, <t>immunoprecipitation;</t> FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.
    Mild Immunoprecipitation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mild immunoprecipitation buffer/product/Thermo Fisher
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mild immunoprecipitation buffer - by Bioz Stars, 2020-03
    88/100 stars
      Buy from Supplier

    99
    Thermo Fisher edta whole blood
    <t>BTV</t> RNA levels in ten animals over a six-month period. Whole <t>(EDTA)</t> blood samples from the first month of virus detection to the last month of blood collection (month 6) were assayed by BTV group-specific rRT-PCRs and Ct values were plotted. Dashed lines indicate those samples that demonstrated gradual increases of Ct values with time; solid lines indicate those with sudden declines in Ct values after initial virus detection.
    Edta Whole Blood, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta whole blood/product/Thermo Fisher
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    edta whole blood - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    77
    Thermo Fisher dialysis edta
    Venn diagrams depicting overlap (i.e., redundancy) of identified proteins between separate fractions. (A) Method 1, (B) Method 2, (C) Method 3, (D) Method 4, (E) Method 5, (F) Method 6, (G) demineralization fractions of Method 7, and (H) a hypothetical method that combines three fractions to maximize recovery. In methods 1–5 (A–E), there was a large amount of overlap between the demineralization and solubilization fractions, and most of the unique proteins were found in the demineralization fractions,rendering the solubilization fractions largely redundant. In Method 6 (F), there were slightly more unique proteins in the two solubilization fractions (combined) than in the one demineralization fraction. In Method 7, there was substantial redundancy in the two sequential demineralization incubations, indicating that additional demineralization did not recover substantially new portions of the proteome. Combining three fractions (i.e., 20-HCl-P, <t>20-N/EDTA-D,</t> and <t>20-H/SDS-P)</t> into a hypothetical extraction (H) accounted for 51 of the 55 proteins identified across all fractions in this study, with the largest contribution of unique proteins coming from HCl.
    Dialysis Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dialysis edta/product/Thermo Fisher
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dialysis edta - by Bioz Stars, 2020-03
    77/100 stars
      Buy from Supplier

    79
    Thermo Fisher edta treated dim 5 protein
    Metal Chelators Inhibit <t>DIM-5</t> Activity (A) Analysis of zinc content of DIM-5 with and without <t>EDTA</t> treatment. DIM-5 protein was incubated with 20 mM EDTA for 2 days, at which time HKMT activity was no longer detectable. To remove zinc bound to EDTA, the protein was either dialyzed (Exp1) or subjected to gel filtration chromatography (Exp2) against 20 mM glycine (pH 9.8), 5% glycerol, 0.5 mM DTT, and 1 mM EDTA. (B) Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1,10-phenanthroline or EDTA for 18 hr at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present. (C) Fluorographic results of AdoMet crosslinking in the presence of EDTA.
    Edta Treated Dim 5 Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta treated dim 5 protein/product/Thermo Fisher
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    edta treated dim 5 protein - by Bioz Stars, 2020-03
    79/100 stars
      Buy from Supplier

    Image Search Results


    Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Plasmid Preparation, Construct, Expressing, Generated, Mutagenesis, Western Blot, Activity Assay, In Vivo, Conjugation Assay, Immunoprecipitation, Quantitation Assay, Two Tailed Test

    Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Liquid Chromatography, Mass Spectrometry, Binding Assay, Immunoprecipitation, SDS Page, Staining, In Vitro, Western Blot, Transfection

    Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Cell Culture, Lysis

    BTV RNA levels in ten animals over a six-month period. Whole (EDTA) blood samples from the first month of virus detection to the last month of blood collection (month 6) were assayed by BTV group-specific rRT-PCRs and Ct values were plotted. Dashed lines indicate those samples that demonstrated gradual increases of Ct values with time; solid lines indicate those with sudden declines in Ct values after initial virus detection.

    Journal: Veterinary Microbiology

    Article Title: Bluetongue virus infection in naïve cattle: Identification of circulating serotypes and associated Culicoides biting midge species in Trinidad

    doi: 10.1016/j.vetmic.2017.09.008

    Figure Lengend Snippet: BTV RNA levels in ten animals over a six-month period. Whole (EDTA) blood samples from the first month of virus detection to the last month of blood collection (month 6) were assayed by BTV group-specific rRT-PCRs and Ct values were plotted. Dashed lines indicate those samples that demonstrated gradual increases of Ct values with time; solid lines indicate those with sudden declines in Ct values after initial virus detection.

    Article Snippet: 2.3 RNA extraction and BTV group- and serotype-specific rRT-PCR Viral RNA was extracted from EDTA whole blood samples at the Non-Vesicular Reference Laboratory (The Pirbright Institute, Surrey, UK) using MagVet Universal Isolation Kits (ThermoFisher Scientific, Paisley, UK) with the KingFisher Flex Purification System (ThermoFisher Scientific, Paisley, UK).

    Techniques:

    Venn diagrams depicting overlap (i.e., redundancy) of identified proteins between separate fractions. (A) Method 1, (B) Method 2, (C) Method 3, (D) Method 4, (E) Method 5, (F) Method 6, (G) demineralization fractions of Method 7, and (H) a hypothetical method that combines three fractions to maximize recovery. In methods 1–5 (A–E), there was a large amount of overlap between the demineralization and solubilization fractions, and most of the unique proteins were found in the demineralization fractions,rendering the solubilization fractions largely redundant. In Method 6 (F), there were slightly more unique proteins in the two solubilization fractions (combined) than in the one demineralization fraction. In Method 7, there was substantial redundancy in the two sequential demineralization incubations, indicating that additional demineralization did not recover substantially new portions of the proteome. Combining three fractions (i.e., 20-HCl-P, 20-N/EDTA-D, and 20-H/SDS-P) into a hypothetical extraction (H) accounted for 51 of the 55 proteins identified across all fractions in this study, with the largest contribution of unique proteins coming from HCl.

    Journal: PeerJ

    Article Title: Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

    doi: 10.7717/peerj.2603

    Figure Lengend Snippet: Venn diagrams depicting overlap (i.e., redundancy) of identified proteins between separate fractions. (A) Method 1, (B) Method 2, (C) Method 3, (D) Method 4, (E) Method 5, (F) Method 6, (G) demineralization fractions of Method 7, and (H) a hypothetical method that combines three fractions to maximize recovery. In methods 1–5 (A–E), there was a large amount of overlap between the demineralization and solubilization fractions, and most of the unique proteins were found in the demineralization fractions,rendering the solubilization fractions largely redundant. In Method 6 (F), there were slightly more unique proteins in the two solubilization fractions (combined) than in the one demineralization fraction. In Method 7, there was substantial redundancy in the two sequential demineralization incubations, indicating that additional demineralization did not recover substantially new portions of the proteome. Combining three fractions (i.e., 20-HCl-P, 20-N/EDTA-D, and 20-H/SDS-P) into a hypothetical extraction (H) accounted for 51 of the 55 proteins identified across all fractions in this study, with the largest contribution of unique proteins coming from HCl.

    Article Snippet: Dialysis EDTA, HCl, SDS, ABC, Urea, GuHCl, and Acetic Acid supernatants were placed into 3,500 MWCO SnakeSkin® dialysis tubing (Thermo Scientific) and dialyzed against 4 L of 18.2 MΩ water (acetic acid fractions were dialyzed against 0.1 M acetic acid) for 4 days at 4 °C, exchanging dialysis water two times daily.

    Techniques:

    Graphs of the total number of (A) peptide spectral matches (PSMs), (B) unique peptides (PTM variations eliminated), and (C) proteins identified within each fraction evaluated by mass spectrometry. (A) The greatest number of PSMs were recovered mainly from demineralization fractions, specifically 20-HCl fractions, 5-HCl-P, and 20-N/EDTA-D fractions, which all recovered 550–450 PSMs. The only solubilization fraction to recover PSMs within this range was 20-H/SDS-P. The next highest values were achieved by 20-H/ABC, whether dialyzed or dried by speed vacuum. This pattern was generally the same for total number of peptides (B), though 5-HCl-P had a greater diversity of unique peptides than higher volume (20-HCl) fractions, which had a greater number of PSMs. This relative pattern was again repeated in the numbers of unique proteins identified (C), which show that HCl, NaOH treated EDTA, and precipitated SDS recovered broader portions of the bone proteome than other extraction steps.

    Journal: PeerJ

    Article Title: Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

    doi: 10.7717/peerj.2603

    Figure Lengend Snippet: Graphs of the total number of (A) peptide spectral matches (PSMs), (B) unique peptides (PTM variations eliminated), and (C) proteins identified within each fraction evaluated by mass spectrometry. (A) The greatest number of PSMs were recovered mainly from demineralization fractions, specifically 20-HCl fractions, 5-HCl-P, and 20-N/EDTA-D fractions, which all recovered 550–450 PSMs. The only solubilization fraction to recover PSMs within this range was 20-H/SDS-P. The next highest values were achieved by 20-H/ABC, whether dialyzed or dried by speed vacuum. This pattern was generally the same for total number of peptides (B), though 5-HCl-P had a greater diversity of unique peptides than higher volume (20-HCl) fractions, which had a greater number of PSMs. This relative pattern was again repeated in the numbers of unique proteins identified (C), which show that HCl, NaOH treated EDTA, and precipitated SDS recovered broader portions of the bone proteome than other extraction steps.

    Article Snippet: Dialysis EDTA, HCl, SDS, ABC, Urea, GuHCl, and Acetic Acid supernatants were placed into 3,500 MWCO SnakeSkin® dialysis tubing (Thermo Scientific) and dialyzed against 4 L of 18.2 MΩ water (acetic acid fractions were dialyzed against 0.1 M acetic acid) for 4 days at 4 °C, exchanging dialysis water two times daily.

    Techniques: Mass Spectrometry

    Metal Chelators Inhibit DIM-5 Activity (A) Analysis of zinc content of DIM-5 with and without EDTA treatment. DIM-5 protein was incubated with 20 mM EDTA for 2 days, at which time HKMT activity was no longer detectable. To remove zinc bound to EDTA, the protein was either dialyzed (Exp1) or subjected to gel filtration chromatography (Exp2) against 20 mM glycine (pH 9.8), 5% glycerol, 0.5 mM DTT, and 1 mM EDTA. (B) Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1,10-phenanthroline or EDTA for 18 hr at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present. (C) Fluorographic results of AdoMet crosslinking in the presence of EDTA.

    Journal: Cell

    Article Title: Structure of the Neurospora SET Domain Protein DIM-5, a Histone H3 Lysine Methyltransferase

    doi:

    Figure Lengend Snippet: Metal Chelators Inhibit DIM-5 Activity (A) Analysis of zinc content of DIM-5 with and without EDTA treatment. DIM-5 protein was incubated with 20 mM EDTA for 2 days, at which time HKMT activity was no longer detectable. To remove zinc bound to EDTA, the protein was either dialyzed (Exp1) or subjected to gel filtration chromatography (Exp2) against 20 mM glycine (pH 9.8), 5% glycerol, 0.5 mM DTT, and 1 mM EDTA. (B) Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1,10-phenanthroline or EDTA for 18 hr at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present. (C) Fluorographic results of AdoMet crosslinking in the presence of EDTA.

    Article Snippet: One sample of untreated and two samples of EDTA-treated DIM-5 protein (about 2 ml of 2 mg/ml each) was analyzed for the presence of 20 elements on a Thermo Jarrell-Ash Enviro 36 ICAP analyzer at the Chemical Analysis Laboratory of the University of Georgia at Athens.

    Techniques: Activity Assay, Incubation, Filtration, Chromatography, Purification, Concentration Assay