We sought to improve MF method 1 given that this method yields insoluble MF aggregates when the pellet is resuspended in the final resuspension buffer (buffer 3). Others have previously demonstrated that the addition of the polyamine spermidine increases protein solubility during tissue processing [30 (link)]. Notwithstanding, it remained to be determined if modifying MF method 1 through the addition of spermidine in the final resuspension solution (buffer 3) would further increase MF protein solubility and, ultimately, protein yield. Procedures for MF method 2 were identical to MF method 1 except that MF pellets were resuspended in 300 μl of ice cold storage buffer as described above (buffer 3: 20 mM Tris-HCl, pH 7.2, 100 mM KCl, 20% glycerol, 1 mM DTT; all chemicals from Ameresco) with the addition of 6.4 M spermidine (Alfa Aesar, Haverhill, MA USA) to yield a working concentration of 50 mM spermidine in Buffer 3.
Spermidine
Manufactured by Thermo Fisher Scientific
Sourced in United States
Spermidine is a naturally occurring polyamine compound found in living cells. It plays a role in various cellular processes, including gene expression, cell growth, and cell differentiation. Spermidine is commonly used as a research tool in laboratories to study these biological functions.
Automatically generated - may contain errors
Lab products found in correlation
Trimethylolpropane triacrylate tmpta, by Merck Group (3 mentions)
1 4 butanediol diacrylate, by Merck Group (3 mentions)
Butylamine, by Merck Group (2 mentions)
Dimethylamine, by Merck Group (2 mentions)
Benzaldehyde, by Thermo Fisher Scientific (2 mentions)
Isoamylamine, by Thermo Fisher Scientific (2 mentions)
Paraffin oil, by Thermo Fisher Scientific (2 mentions)
Cadaverine, by Merck Group (2 mentions)
Benzaldehyde, by Merck Group (2 mentions)
Ammonium hydroxide, by Thermo Fisher Scientific (2 mentions)
Ethylamine, by Merck Group (2 mentions)
Trimethylamine, by Merck Group (2 mentions)
Putrescine, by Thermo Fisher Scientific (1 mentions)
Spermine, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Aminoguanidine, by Merck Group (1 mentions)
Fibrillarin, by Cell Signaling Technology (1 mentions)
Anti rabbit igg alexa488, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
8 protocols using spermidine
1
Improving Membrane Fractionation Protein Yield
2
Odorant Preparation and Concentrations
3
Chemically Defined Medium for Streptococcus agalactiae
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The liquid chemically defined medium used to grow S. agalactiae d,l -alanine, 757 µM l -asparagine, 1127 µM l -aspartic acid, 684.5 µM l -glutamine, 1019.5 µM l -glutamic acid, 868.6 µM l -proline, 734.9 µM l -tryptophan, 4125.4 µM l -cysteine, 12 µM lipoic acid, 1 µM pyruvate, 17.4 µM ZnSO4.7H2O, 10.5 µM CoCl2.H2O, 0.4 µM CuSO4.5H2O and 55 mM d -glucose. This medium was finally adjusted to pH 7.4 with HCl. For some experiments, CDM was buffered at pH 7.4 with 100 mM HEPES (Fisher Bioreagents; cat. no. BP310-100), to pH 5.5 with 100 mM MES (ACROS Organics; code no. 172591000) or pH 4.0 by the addition of 33.5 mM sodium citrate dihydrate and 66.5 mM citric acid. Final adjustments of the pH were made with citric acid or HCl. Spermidine (ACROS Organics; Cat. No. AC132740050), putrescine (ACROS Organics; Cat. No. AC132750050) or spermine (ACROS Organics; Cat. No. AC112120250) were also added for some experiments.
4
Polyamine Transport Inhibitor Synthesis
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DFMO was kindly provided by Dr. Patrick Woster (Medical University of South Carolina, Charleston, SC). The trimer44NMe polyamine transport inhibitor (PTI) and the N1-(4-nitro-benzo-[1,2,5] -oxadiazolyl) spermine derivative were synthesized in house8 (link),27 (link). Aminoguanidine was purchased from Sigma-Aldrich. Spermidine was acquired from Acros Organics and 3H-Spd and 3H-Put were purchased from Perkin-Elmer Inc. 14C-Spm was obtained as a gift from Dr. Tracy Murray-Stewart (Johns Hopkins). Primary antibodies for ATP13A3 (HPA029471) and β-actin were obtained from Sigma. Fibrillarin and GRASP65 antibodies were obtained from Cell Signaling Technology, and the anti-SLC12A8 antibody was custom synthesized by GenScript. Anti-mouse and anti-rabbit secondary antibodies for immunoblotting were purchased from Santa Cruz Biotechnology and anti-mouse IgG Alexa568 and anti-rabbit IgG Alexa488 were obtained from Invitrogen.
5
Odorant Preparation and Dilution Protocol
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Odorants were ammonium hydroxide (Fisher or VWR International), trimethylamine (Sigma), dimethylamine (Sigma), ethylamine (Sigma), butylamine (Sigma), cadaverine (Sigma), spermidine (ACROS), isoamylamine (ACROS), and benzaldehyde (Sigma). All odorants were diluted to generate 10% stocks, and serial dilutions were used to generate lower concentrations.
All odorants were diluted in water, except benzaldehyde, which was diluted in paraffin oil (ACROS). Ammonia was used at concentrations from 0.0001% to 1% for electrophysiology and transcuticular imaging. Other odorants used for electrophysiological experiments were at 1% concentration. All odorants were used at a 0.01% concentration for antennal lobe GCaMP imaging. For behavioral assays, ammonia was used at 0.3% and benzaldehyde at 1%.
All odorants were diluted in water, except benzaldehyde, which was diluted in paraffin oil (ACROS). Ammonia was used at concentrations from 0.0001% to 1% for electrophysiology and transcuticular imaging. Other odorants used for electrophysiological experiments were at 1% concentration. All odorants were used at a 0.01% concentration for antennal lobe GCaMP imaging. For behavioral assays, ammonia was used at 0.3% and benzaldehyde at 1%.
6
Naturally Occurring Amine-Containing Cement
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Example 32
The following example provides a cement product of the invention that comprises a second component with a naturally occurring amine.
In a 20 ml vial, 1.3294 gm of 1,4-butanedioldiacrylate from Aldrich, was added with 0.6638 gm trimethylolpropane triacrylate (TMPTA) from Sigma-Aldrich. The mixture was stirred for 30 second, it was then reacted with 0.526 gm of spermidine from Alpha Aesar (Ward Hill, Mass.), and the reaction was stirred to homogenous mixture within 1 minute. The temperature rose from 25° C. to 57° C. in 2 minute. The working time was 3 minutes and the neat cement set to dried to touch in 5 minutes.
7
Naturally Occurring Amine-Based Cement Formulation
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Example 32
The following example provides a cement product of the invention that comprises a second component with a naturally occurring amine.
In a 20 ml vial, 1.3294 gm of 1,4-butanedioldiacrylate from Aldrich, was added with 0.6638 gm trimethylolpropane triacrylate (TMPTA) from Sigma-Aldrich. The mixture was stirred for 30 second, it was then reacted with 0.526 gm of spermidine from Alpha Aesar (Ward Hill, Mass.), and the reaction was stirred to homogenous mixture within 1 minute. The temperature rose from 25′ C to 57° C. in 2 minute. The working time was 3 minutes and the neat cement set to dried to touch in 5 minutes.
8
Naturally Occurring Amine Cement Formulation
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Example 32
The following example provides a cement product of the invention that comprises a second component with a naturally occurring amine.
In a 20 ml vial, 13294 gm of 1,4-butanedioldiacrylate from Aldrich, was added with 0.6638 gm trimethylolpropane triacrylate (TMPTA) from Sigma-Aldrich. The mixture was stirred for 30 second, it was then reacted with 0.526 gm of spermidine from Alpha Aesar (Ward Hill, Mass.), and the reaction was stirred to homogenous mixture within 1 minute. The temperature rose from 25° C. to 57° C. in 2 minute. The working time was 3 minutes and the neat cement set to dried to touch in 5 minutes.
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