Cell pellets from 1 L LB culture were suspended with 30 mL of buffer A (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 20 mM imidazole, 5 mM beta-mercaptoethanol (BME), 1% Tween20) supplemented with a tablet protease inhibitor cocktail (complete, EDTA-Free, Sigma). Cells were lysed by sonication. Soluble fractions were separated by centrifugation (20,000 × g for 40 min at 4 °C). The supernatant was loaded on a pre-equilibrated 1 mL HisTrap column (GE Healthcare, USA) with buffer B (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 20 mM imidazole) and washed with 10 column volumes (CV) of buffer B and 50 CV of buffer C (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 50 mM imidazole), and eluted with buffer D (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 300 mM imidazole). Eluent was injected to HiLoad 26/600 superdex 200 pg (GE Healthcare, USA) column equilibrated with buffer E (20 mM HEPES pH 7.5, 300 mM NaCl, 10% Glycerol, 0.5 mM TCEP). The major fractions were analyzed with 4–20% SDS-PAGE Gel stained with coomassie brilliant blue. Protein concentrations were determined by Bradford protein assay kit II (Bio-Rad, USA) with bovine serum albumin as the standard.
Hiload 26 600 superdex200 pg
Manufactured by GE Healthcare
Sourced in United States
The HiLoad 26/600 Superdex200 PG is a size-exclusion chromatography column designed for protein purification. It is a prepacked column with a bed volume of 320 mL and a matrix composition of cross-linked agarose and dextran.
Automatically generated - may contain errors
Lab products found in correlation
Akta purifier 10, by GE Healthcare (8 mentions)
Histrap column, by GE Healthcare (4 mentions)
Complete edta free, by Merck Group (1 mentions)
Bradford protein assay kit 2, by Bio-Rad (1 mentions)
Sp sepharose fast flow, by GE Healthcare (1 mentions)
Bl21 codonplus de3 rp, by Agilent Technologies (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
Sephadex g 25 column, by GE Healthcare (1 mentions)
Chelating sepharose, by GE Healthcare (1 mentions)
Nupage 10 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Kta avant 25, by GE Healthcare (1 mentions)
Syringe filter, by Merck Group (1 mentions)
Hitrap q hp 5 ml column, by GE Healthcare (1 mentions)
Sigmafast protease inhibitor, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Biliverdin hydrochloride, by Frontier Scientific (1 mentions)
Amicon ultracel 10k, by Merck Group (1 mentions)
M 110l microfluidizer, by Microfluidics (1 mentions)
19 protocols using hiload 26 600 superdex200 pg
1
Purification of His-Tagged Proteins
2
Purification of Recombinant Tau and α-Synuclein
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Escherichia coli BL21-CodonPlus (DE3)-RP (Agilent) was transformed with a pET28a plasmid encoding WT or P301L tau (full-length 0N4R). Terrific broth cultures (1 l) supplemented with 50 mg/l kanamycin or 50 mg/l chloramphenicol were inoculated with 20 ml of starter cultures and grown for 8 h. The cultures were induced with 1 mM IPTG and grown for another 16 h. Cells were harvested and resuspended in 50 ml/l 20 mM MES, pH 6.8, 1 mM EGTA, 1 mM magnesium chloride, 5 mM DTT, and 1 cOmplete protease inhibitor cocktail (Roche) followed by microfluidizer lysis. The lysates were boiled for 20 min and centrifuged at 48,400g. The cleared lysates were applied to a cation exchange column (SP Sepharose Fast Flow; GE Healthcare), and fractions were eluted with a sodium chloride gradient. Fractions containing 0N4R tau were applied to a reversed-phase HPLC column and eluted with an acetonitrile gradient (1%/min) + 0.1% TFA gradient; the peak fractions were then lyophilized. The lyophilizates were dissolved in PBS + 1 mM DTT and purified by size-exclusion chromatography (HiLoad 26/600 Superdex 200 pg; GE Healthcare). Peak fractions were analyzed by SDS-PAGE, and fractions containing <95% 0N4R tau were pooled, snap-frozen, and stored at −80 °C. Recombinant full-length α-synuclein and tau repeat domain (K18) were expressed and purified as previously described (72 (link), 73 (link)).
3
Trastuzumab Bioconjugate Preparation
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Example 28
A bioconjugate according to the invention was prepared by conjugation of compound 78 as linker-conjugate to azide-modified trastuzumab as biomolecule. To a solution of trastuzumab-(6-N3-GalNAc)2 (13d) (8.18 mL, 270 mg, 33.0 mg/ml in PBS pH 7.4) was added PBS pH 7.4 (7.12 mL), DMF (2.48 mL) and compound 78 (225 μL, 40 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a HiLoad 26/600 Superdex200 PG (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25921 Da, approximately 90% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment. RP-HPLC analysis of the reduced sample indicated an average DAR of 1.90.
4
Purification of MBP-Cit1 and MBP-Cit2
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To purify MBP-Cit1 and MBP-Cit2 for assay, frozen cell pellets were thawed and resuspended in lysis buffer. Subsequently, the cells were lysed by sonication, and cell debris was removed by centrifugation. The supernatant was subjected to Amylose resin affinity column chromatography (Amylose resin, NEB). Unbound proteins were washed away using lysis buffer, and then the bound protein was eluted from the column using amylose column elution buffer [20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 10 mM maltose]. Proteins were purified further by size exclusion column chromatography (HiLoad 26/600 Superdex 200 pg, GE HealthCare) using an FPLC system. The column was equilibrated and eluted with size exclusion column buffer A. The eluates were dialyzed overnight at 4°C against dialysis buffer and then concentrated to ~3 mg/ml using a centrifugal concentrator.
5
Recombinant LAMP1 and mAb Production
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Nucleic acid sequences coding for LAMP1 extracellular domains fused to his-tag or Halotag-histag at the C-terminal or coding for the antibody heavy or light chains were cloned into mammalian expression plasmids under the CMV enhancer/promoter and the SV40 polyA signal. Resulting plasmids were transfected into HEK293 cells (Thermo Fisher Scientific; K9000-10) using FreeStyle™ MAX 293 Expression System according to the manufacturer’s instructions. LAMP1 proteins were purified by immobilized metal affinity chromatography (Chelating Sepharose, 17-0575-01 GE Healthcare) and stored in PBS after concentration and buffer exchange (Sephadex G-25 column, GE Healthcare).
For initial characterization, mAbs were produced at 30 mL scale, purified by protein A affinity chromatography and stored in PBS after desalting on mini trap Sephadex G-25 column. For the developability study, mAbs were produced at 1 L scale and purified by a two-step process including protein A affinity chromatography (HiScreen MabSelect Sure protein A, GE Healthcare, 28-9269-77) and size-exclusion chromatography (HiLoad 26/600 superdex 200 pg, GE Healthcare, 28-9893-36).
For initial characterization, mAbs were produced at 30 mL scale, purified by protein A affinity chromatography and stored in PBS after desalting on mini trap Sephadex G-25 column. For the developability study, mAbs were produced at 1 L scale and purified by a two-step process including protein A affinity chromatography (HiScreen MabSelect Sure protein A, GE Healthcare, 28-9269-77) and size-exclusion chromatography (HiLoad 26/600 superdex 200 pg, GE Healthcare, 28-9893-36).
6
Protein Purification by Size-Exclusion Chromatography
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The protein component was filtered through a 0.45-μm filter membrane before purification. ÄKTA Avant 25 (GE Healthcare, WA, United States) equipped with HiLoad 26/600 Superdex 200 pg (GE Healthcare) was used for size-exclusion chromatography. The protein component was loaded at the rate of 5 ml/min, and 20 mm Tris, pH 5.0 was used for elution at the rate of 3 ml/min. The eluates corresponding to different signal peaks were collected. Amicon Ultra Centrifugal Filters of 10k MWCO (Merck, NJ, United States) were used for the protein concentration at 3000 g and 4°C until the volume was concentrated to 200 μl. SDS-PAGE was used for separating the protein concentrates using a NuPAGE 10% Bis-Tris Gel (Thermo Fisher Scientific, MA, United States). The protein bands were excised from the gel. The protein profiles provided by Sangon (Shanghai, China) were used for identifying the protein bands.
7
Bioconjugation of Compound 63 to cAC10
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Example 35
A bioconjugate according to the invention was prepared by conjugation of compound 63 as linker-conjugate to azide-modified cAC10 as biomolecule. To a solution of cAC10-(6-N3-GalNAc)2 (13d) (9.95 mL, 205 mg, 20.7 mg/ml in PBS pH 7.4) was added PBS pH 7.4 (1.0 mL), DMF (2.568 mL) and compound 63 (171.7 μL, 40 mM solution in DMF). The reaction was incubated at rt overnight followed by dialysis and purification on a HiLoad 26/600 Superdex200 PG (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 27124 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment. RP-HPLC analysis of the reduced sample indicated an average DAR of 3.79.
8
Synthesis of Bioconjugate from Compound 66
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Example 34
A bioconjugate according to the invention was prepared by conjugation of compound 66 as linker-conjugate to azide-modified cAC10 as biomolecule. To a solution of cAC10-(6-N3-GalNAc)2 (13d) (8.408 mL, 246.0 mg, 29.3 mg/ml in PBS pH 7.4) was added propylene glycol (11.909 mL) and compound 66 (410.6 μL, 40 mM solution in DMF). The reaction was incubated at rt for approximately 40 hrs. The reaction mixture was dialyzed to PBS pH 7.4 and purified on a HiLoad 26/600 Superdex200 PG (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 27132 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment. RP-HPLC analysis of the reduced sample indicated an average DAR of 3.81.
9
Site-Specific Trastuzumab Bioconjugation
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Example 31
A bioconjugate according to the invention was prepared by conjugation of compound 73 as linker-conjugate to azide-modified trastuzumab as biomolecule. To a solution of trastuzumab-(6-N3-GalNAc)2 (13d) (4.09 mL, 135 mg, 33.0 mg/ml in PBS pH 7.4) was added PBS pH 7.4 (2.66 mL), DMF (2.14 mL) and compound 73 (113 μL, 40 mM solution in DMF). The reaction was incubated at rt overnight followed by dialysis against PBS pH 7.4 (0.5 L) and purification on a HiLoad 26/600 Superdex200 PG (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 227331 Da, approximately 90% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment. RP-HPLC analysis of the reduced sample indicated an average DAR of 3.78.
10
Bioconjugation of Trastuzumab with Compound 44
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Example 64
A bioconjugate according to the invention was prepared by conjugation of compound 44 as linker-conjugate to azide-modified trastuzumab as biomolecule. To a solution of trastuzumab-(6-N3-GalNAc)2 (13d) (4.239 mL, 120 mg, 28.31 mg/ml in PBS pH 7.4) was added PBS pH 7.4 (1.761 mL), DMF (1.680 mL) and compound 44 (0.320 mL, 10 mM solution in DMF). The reaction was incubated at rt overnight followed by purification on a HiLoad 26/600 Superdex200 PG (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 27398 Da, approximately 90% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment. RP-HPLC analysis of the reduced sample indicated an average DAR of 3.77.
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