Proteins can be electrokinetically extracted from single plaques for microanalysis by mass spectrometry and amino acid analysis, but these quantities are insufficient to see on a gel or to coacervate. For gels, plaques (<24 hours old) were collected and pooled for bulk homogenization at a ratio of ~50-mg wet weight to 1 ml of either 0.7% acetic acid (pH 3) or 121 mM tris-glycinate (pH 8.2) to match electrotransfer conditions. The homogenate was clarified via centrifugation at 5000g for 10 min at which point the supernatant solution was carefully separated from the insoluble fraction. The pH 8.2 homogenate was then dialyzed against 0.7% acetic acid for >4 hours at 4°C. Both homogenates were mixed 1:1 with gel-loading buffer (5% acetic acid and 8 M urea) (32 (link)), and 10 μl was loaded onto a preequilibrated 7.5% acid-urea gel (10 mA for 2 hours). The gel was run with 5% acetic acid, serving as the tank buffer in a Bio-Rad Mini-PROTEAN Tetra Cell (Hercules, CA) at a constant current of 12 mA for 30 min using a GE EPS 301 power supply. After electrophoresis, the gel was stained with 0.1% Coomassie Brilliant Blue (CBB) G-250 in 40% methanol and 20% acetic acid for 30 min and destained with 20% methanol and 10% acetic acid until the gel background became completely transparent.
Eps 301
Manufactured by GE Healthcare
Sourced in United States
The EPS 301 is a piece of lab equipment manufactured by GE Healthcare. It is a compact and versatile electrophoresis system designed for the separation and analysis of biomolecules, such as proteins and nucleic acids. The EPS 301 provides a reliable and consistent performance for researchers and scientists working in various fields of biology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Milli q reference, by Merck Group (3 mentions)
Elix uv, by Merck Group (3 mentions)
Eps 601, by GE Healthcare (3 mentions)
Novex ph 3 10, by Thermo Fisher Scientific (1 mentions)
Hi qras gel n, by Kanto Chemical (1 mentions)
J 820, by Jasco (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Microfuge 16 centrifuge, by Beckman Coulter (1 mentions)
L glycine, by Fujifilm (1 mentions)
6 protocols using eps 301
1
Extracting Proteins from Plaques for Mass Spectrometry
2
Characterization of HSA-Hb Conjugation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human serum albumin (HSA) was purchased from the Japan Blood Products Organization. Hemoglobin (Hb) was purified from bovine red blood cells purchased from Tokyo Shibaura Zouki Co. Ltd. Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was purchased from Wako Pure Chemical Industries Ltd. 1,11-Bismaleimido-triethyleneglycol (BMTEG) was purchased from Thermo Fischer Scientific K.K. The water was deionized (18.2 MΩcm) using water purification systems (Elix UV and Milli Q Reference; Millipore Corp.). Native-PAGE and SDS-PAGE were conducted using an electrophoresis power supply (EPS 301; GE Healthcare) with precast 5‒20% polyacrylamide gel (Hi-QRAS Gel N 5‒20%: Kanto Chemical Co. Inc.). Isoelectric focusing (IEF) was performed using an electrophoresis power supply (EPS 601; GE Healthcare) with a pH 3–10 IEF protein gel (Novex; Thermo Fischer Scientific K.K.). The voltage was raised gradually to 500 V for 2 h.
3
Purification and Characterization of Bovine Hemoglobin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bovine Hb was purified from fresh bovine blood purchased from Tokyo Shibaura Zouki Co. Ltd. Purification procedures of native CSA from canine plasma are presented in Supplementary Materials . All other reagents were purchased from commercial sources as special grades and were used without further purification. Deionized water (18.2 MΩ·cm) was prepared using water purification systems (Elix UV and Milli Q Reference; Millipore Corp.). SDS-PAGE and Native-PAGE were performed using an electrophoresis power supply (EPS 301; GE Healthcare UK Ltd.) and 5–20% polyacrylamide precast gradient gel (Hi-QRAS gel N; Kanto Chemical Co. Inc.). Isoelectric focusing (IEF) was conducted using an electrophoresis power supply (EPS 601; GE Healthcare UK Ltd.) and IEF protein gels (Novex pH 3–10; Thermo Fisher Scientific Inc.). The UV-visible absorption spectra were recorded using a UV-visible spectrophotometer (8543; Agilent Technologies Inc.) connected with a temperature control unit (89090A; Agilent Technologies Inc.). Circulation dichroism (CD) spectra were recorded using a spectrophotometer (J-820; Jasco Corp.). Mass spectra were obtained using a MALDI–TOF mass spectrometer (autoflex equipped with a pulsed N2 laser (337 nm); Bruker Daltonics K.K.). As the matrix, 0.1% sinapic acid, 0.05% trifluoroacetic acid, and 50% acetonitrile solution were used.
4
Mitochondrial RNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mitochondrial RNA was isolated from myocardial tissues using Trizol (Invitrogen Inc., Carlsbad, CA, USA), and then reverse-transcribed using the RT-PCR ReverTra-PlusTM kit (TOYOBO Co., Ltd, Tokyo, Japan) according to the manufacturer’s instructions. Semi-quantitative RT-PCR analysis of COX I and COX III mRNA expressions was performed using the Prime Script RT-PCR kit (Clontech Laboratories Inc., Mountain View, CA, USA) according to the manufacturer’s instructions and a PCR system (AG-22331; Eppendorf, Hamburg, Germany). Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as control. The primers were: COX I: forward: 5′-GGC TTC GGG AAC TGA CTT GT-3′, reverse: 5′-AAG GAT TGG GTC TCC ACC TC-3′ (annealing temperature: 60 °C; 30 cycles; PCR product: 462 bp); COX III: forward: 5′-GCC ACC ACA CCC CTA TTG TA-3′, reverse: 5′-TCC CGT TGC TAT GAA GAA TG-3′ (annealing temperature: 60 °C; 30 cycles; PCR product: 401 bp), and GAPDH: forward: 5′-TGT TCC TAC CCC CAA TGT GT-3′, reverse: 5′-CCC TGT TGC TGT AGC CGT AT-3′ (annealing temperature: 60 °C; 30 cycles; PCR product: 401 bp). The relative expression of mRNA of a specific gene to the internal control was calculated as the ratio of relative optical density using a gel imaging analyzer (EPS301; GE Healthcare, Waukesha, WI, USA) with gel analysis software (GE Healthcare, Waukesha, WI, USA).
5
Molecular Techniques for Biomedical Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KS12 biological safety cabinet (Thermo Fisher Scientific, Dreieich, Germany); Lab Dancer (IKA, Königswinter, Germany); Microfuge® 16 centrifuge (BECKMAN, Brea, CA, USA); DK-S26 electric thermostatic water bath (Shanghai Senxin Experimental Instrument Co., Ltd., Shanghai, China); Nanodrop 2000 (ThermoFisher Scientific, Germany); Biometra Tadvanced 96 SG (Biometra, Göttingen, Germany); Lab cycler Gradient (SensoQuest, Göttingen, Germany); CFX96 Real-Time PCR system (Bio-RAD, Hercules, CA, USA); Automatic Gel Imaging analysis SystemZF-258 (Shanghai Jiapeng Technology Co., Ltd., Shanghai, China); Electrophoresis power supply EPS 301 (GE, Boston, MA, USA).
6
Characterization of Modified Human Serum Albumin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was purchased from Tokyo Chemical Industry Co. Ltd. Human serum albumin (HSA) was purchased from the Japan Blood Products Organization. Bis(3,5-dibromosalicyl)fumarate (DBBF) was purchased from LKT Laboratories Inc. L-Glycine was purchased from Wako Pure Chemical Industries Ltd. Inositol hexaphosphate (IHP) was purchased from Sigma-Aldrich Corp. Human adult Hb (HbA) was purified from red blood cell concentrates received from the Japanese Red Cross Society (see ESI †). Other chemicals of special grade were used without further purification unless otherwise noted. Water was deionized (18.2 MO cm) using water purification systems (Elix UV and Milli Q Reference; Millipore Corp.). Native-PAGE was performed using an electrophoresis power supply (EPS 301; GE Healthcare) using 5-12% polyacrylamide precast gradient gel (SuperSep Ace 5-12%; Wako Pure Chemical Industries Ltd). Isoelectric focusing (IEF) was done using an electrophoresis power supply (EPS 601; GE Healthcare) using a pH 3-10 IEF gel (Novex; Invitrogen Corp.). A GE Healthcare IEF calibration kit Broad pI (pH 3-10) was used for the protein marker. The voltage was raised gradually to 500 V for 2 h.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!