Aβ protein was prepared using a protocol known to produce low-weight oligomers78 (link). To confirm the success and quality of our oligomerization protocol, gel electrophoresis of pure Aβ protein was performed, followed by a coomassie blue stain. Mini-PROTEAN® 10–20% Tris-Tricine Precast Gels (Bio-Rad, 456–3113) were loaded with the purified Aβ peptide suspended in Tricine Sample Buffer (Bio-Rad, #161–0739), and electrophoresed for 1–2 hrs at 120 V in Tris-Tricine SDS buffer (Sigma-Aldrich, T1165). Gels were then stained with Coomassie Brilliant Blue (Bio-Rad, 161-0786), and destained (Bio-Rad, Coomassie Brilliant Blue R-250 Destaining Solution 161-0438161-0438) to evaluate low-weight oligomer formation (Supplemental Figure 2 ).
Coomassie brilliant blue r 250 destaining solution
Manufactured by Bio-Rad
Sourced in Germany
Coomassie Brilliant Blue R-250 Destaining Solution is a laboratory reagent used to remove excess Coomassie Brilliant Blue R-250 dye from stained protein gels. The solution facilitates the destaining process, allowing for clear visualization of protein bands after electrophoretic separation.
Automatically generated - may contain errors
Lab products found in correlation
Coomassie brilliant blue r 250 staining solution, by Bio-Rad (5 mentions)
Tris hcl precast gel, by Bio-Rad (2 mentions)
Odyssey clx imager, by LI COR (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Tricine sample buffer, by Bio-Rad (1 mentions)
Mini protean 10 20 tris tricine precast gels, by Bio-Rad (1 mentions)
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Bovine hb, by Merck Group (1 mentions)
Ge 2d quant kit, by GE Healthcare (1 mentions)
Precision plus protein dual xtra standard, by Bio-Rad (1 mentions)
Pepsin a, by Worthington (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Faramount, by Agilent Technologies (1 mentions)
Las 4000 mini, by GE Healthcare (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
Ready gel tris hcl gel, by Bio-Rad (1 mentions)
Synergy h1 plate reader, by Agilent Technologies (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
8 protocols using coomassie brilliant blue r 250 destaining solution
1
Characterization of Aβ Oligomers via Gel Electrophoresis
2
Plasma Albumin Quantification by SDS-PAGE
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Albumin deficiency was previously demonstrated in Alb−/− mice through mass spectrometry analysis [17 ]. To confirm this expectation in the present study, SDS-PAGE was used. Plasma samples were diluted with gel loading buffer containing Laemmli Sample Buffer (Biorad, Hercules, CA, USA) and β-mercaptoethanol. Plasma proteins were then separated by 4–15% Tris–HCl precast gel (BioRad), with the human albumin standard as a reference. After that, the gel was stained by Coomassie Brilliant Blue R-250 Staining Solution (Biorad) for one hour, followed by destaining with Coomassie Brilliant Blue R-250 Destaining Solution (Biorad). The resulting gel was then scanned using an Odyssey CLx Imager (LI-COR Biosciences, Lincoln NE, USA).
3
Pepsin Digestion of Soy LegHb Protein
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The protein sample solution was provided by Impossible Foods Inc. from the LegHb production run PP‐PGM2‐15‐320‐101. The LegHb protein represented 66% of the total protein according to the certificate of analysis. The total protein concentration was determined to be 79.94 mg mL–1 using a GE 2D Quant kit (GE Healthcare, #80‐6483‐56), following the kit instructions. The PP‐PGM2‐15‐320‐101 is a representative batch of soy LegHb protein preparation. The type and abundance of Pichia proteins is consistent from batch‐to‐batch. Bovine Hb (Sigma–Aldrich Co. LLC., St. Louis, MO, #H2625‐25G), BSA (Sigma‐Aldrich Co. LLC., MO #A9647‐100G), and chicken ovalbumin (OVA; Worthington Biochemicals, #3054) were used as control proteins in the digestion assay.
Pepsin A (Worthington Biochemicals, #3319) with a certificate of analysis of 2810 activity units per mg solid was used in all assays. Novex® 10–20% tris‐glycine polyacrylamide gels (Invitrogen) were used to separate digested materials. Precision Plus ProteinTM dual xtra standards (BioRad) were used as the molecular weight standards. Tris‐Glycine‐SDS 10 × running buffer (Fisher Scientific), Coomassie Brilliant Blue R‐250 staining solution, and Coomassie Brilliant Blue R‐250 destaining solution (BioRad) were used for gel running, staining, and destaining, respectively.
Pepsin A (Worthington Biochemicals, #3319) with a certificate of analysis of 2810 activity units per mg solid was used in all assays. Novex® 10–20% tris‐glycine polyacrylamide gels (Invitrogen) were used to separate digested materials. Precision Plus ProteinTM dual xtra standards (BioRad) were used as the molecular weight standards. Tris‐Glycine‐SDS 10 × running buffer (Fisher Scientific), Coomassie Brilliant Blue R‐250 staining solution, and Coomassie Brilliant Blue R‐250 destaining solution (BioRad) were used for gel running, staining, and destaining, respectively.
4
Microscopic Evaluation of Cell Morphology and Silicone Oil Uptake
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For light microscopic evaluation of morphology and silicone oil uptake, cells were stained with Coomassie. After incubation and removal of the supernatant, cells were washed with PBS and fixed for 40 min with 2.5% glutaraldehyde (Merck, Darmstadt, Germany). Cells were washed with PBS and stained for 10 min with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad, München, Germany) and destained for 2 × 20 min with Coomassie Brilliant Blue R-250 destaining solution (Bio-Rad). Afterwards, cells were washed three times with PBS and once with Aqua Dest (Ampuwa, Fresenius, Bad Homburg, Germany) and mounted with Faramount (Dako, Hamburg, Germany).
5
Gelatin Zymography for MMP-9 Activity
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Cell culture supernatants were resolved under nonreducing conditions by SDS-PAGE (10% wt/vol) polymerized in the presence of gelatin (0.1% wt/wt) to evaluate the activities of MMP-9. Gels were washed with Triton X-100 (2.5% vol/vol) for 60 min and distilled water for 15 min two times. Gels were then incubated overnight at 37°C in 50 mM Tris buffer containing calcium chloride (5 mM) pH 8.0. After incubation, gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad, Cat. No. 161-0436) for 30 min at room temperature, then followed by destaining with Coomassie Brilliant Blue R-250 Destaining Solution (Bio-Rad, Cat. No. 161-0438) until clear bands appeared. Gel images were quantified using a General Electric Imager (GE Healthcare, LAS 4000 mini); the unstained, translucent digested regions represented areas of MMP activity [30 (link)].
6
SDS-PAGE Protein Quantification and Visualization
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SDS-PAGE was carried out according to the stacking gel procedure as described by Laemmli (1970) (link). Protein concentration was quantified using a Synergy H1 plate reader (Bio-Tek Instruments, Inc., USA) with the take microdrop addition. Each sample was redissolved in SDSPAGE sample buffer [62.5 mM Tris-HCl, pH 6.8; 2% (w/v) SDS; 25% (v/v) glycerol; 5% (v/v) 2-mercaptoethanol; 0.01% (w/v) bromophenol blue] and denatured at 100℃ for 5 min. Twenty micrograms of the samples were loaded on 10% Ready Gel (Tris-HCl Gel, Bio-Rad, Hercules, USA). The equipment employed was the Mini-PROTEAN® Tetra Cell (Bio-Rad). The gels were stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio- Rad). Destaining was carried out with a Coomassie Brilliant Blue R-250 Destaining Solution (Bio-Rad).
7
Coomassie Brilliant Blue Staining and Destaining
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Gels were incubated for at least one hour in Coomassie (40% (v/v) methanol, 10% (v/v) acetic acid, 0.25% (m/v) Coomassie blue R250) and then destained for at least two hours in destaining solution (40% (v/v) methanol, 10% (v/v) acetic acid).
For mass spectrometry analyses, gels were stained with Brilliant Blue R-250 Coomassie (BioRad) for at least one hour and then destained with Coomassie Brilliant Blue R-250 Destaining Solution (BioRad) for at least two hours.
For mass spectrometry analyses, gels were stained with Brilliant Blue R-250 Coomassie (BioRad) for at least one hour and then destained with Coomassie Brilliant Blue R-250 Destaining Solution (BioRad) for at least two hours.
8
Plasma Protein Separation by SDS-PAGE
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Plasma samples were diluted 40-fold with Laemmli Sample Buffer (Biorad, Hercules, CA, USA) and β-mercaptoethanol. 20 μL of diluted plasma were then loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–15% Tris-HCl precast gel (BioRad), with human serum albumin as a reference. The gel electrophoresis was conducted with a voltage set at 100 V for 10 min and at 200 V for approximately 45 min until the loading dye reached near the bottom of the gel. The gel was then stained by Coomassie Brilliant Blue R-250 Staining Solution (Biorad) for one hour, followed by destaining with Coomassie Brilliant Blue R-250 Destaining Solution (Biorad). The resulting gel was then scanned using an Odyssey CLx Imager (LI-COR Biosciences, Lincoln NE, USA).
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