Superdex 75 Increase 10/300 (blue), Superdex 0 Increase 10/300 (green) , Elution volume (ml) Superdex 0 Increase Superose 6 Increase Superdex 75 Increase 17 21 23 60 50 40 30 10 0 0 25 50 75 Time (min) A 280nm (mAU) A 280nm (mAU) Time (min) 100 125 150 170 160 150 140 1 110 100 90 80 70 60 50 40 30 10 0 0 10 30 40Packing instructions Superdex Prep Grade Column Slurry Slurry Compression Packed bed Flow rate Time for concentravolume % 15 height (ml) rate step 1 035 packing tion % 60 – 2 (ml) (ml/min) (ml/min) step 1 (min) XK 16/100 52 – 2 step 2 (MPa) 430 93–94 3 –05 90 XK 26/100 1000 10 4 –05 90 XK 50/100 65–70 3000 94–95 10 –2Superdex 75 10/300 GL and Superdex 0 10/300 GL are Tricorn™ high performance columns The columns are prepacked glass columns for high performance gel filtration of proteins, peptides, DNA fragments (
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Superdex 75 16/60 void volume
Superdex 75 16/60 void volume-Superdex 75 Increase 10/300 GL is a versatile, prepacked column for high resolution size exclusion chromatography (SEC) in small scale (25 500 µl sample volume) The column is intended for use in preparative purification as well as for characterization and analysis of proteins with molecularV0 void volume Vc geometric column volume Ve elution volume Vt total liquid volume HiLoad 16/600 Superdex 75 pg 3 × 10to 7 × 103 4 1 500 HiPrep 16/60 Sephacryl S0 HR 5 × 10to 25 × 103 5 1 500 HiPrep 26/60 Sephacryl S0 HR 5 × 10to 25 × 103 5 3 1000
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0 = void volume V c = geometric column volume V e = elution volume V t = total liquid volume 6 4 Introduction Two Gel Filtration Calibration Kits are available for protein HiLoad 16/60 Superdex 75 pg 3 × 103 to 7 × 104 1 500 HiLoad 26/60 Superdex 75 pg 3Superdex ® 75 10/300 GL is a prepacked gel filtration column for highresolution, semipreparative, and analytical separations of biomolecules Superdex ® 75 has a separation range for molecules with molecular weights between 3 000 and 70 000 Superdex ® prepacked columns are designed for highperformance, laboratoryscale separations of proteins, peptides, and otherSelectivity curves from Superdex 30 prep grade, Superdex 75 prep grade, and Superdex 0 prep grade Column HiLoad 26/600 Superdex 30 pg Sample Mix of five synthetic peptides in 1% TFA 1 M r 34 2 M r 3134 3 M r 2365 4 M r 1596 5 M r 7 Sample volume 50 μL Buffer mM TrisHCl, 025 M NaCl, pH 85 Flow rate 1 mL/min (30 cm/h
• Blue Dextran 00 to determine the void fraction in the column by comparing an elution volume parameter, such as the gel phase distribution coeffi cient (Kav) of the protein of interest, proteins on HiLoad 16/60 Superdex 75 pg column Fig 7Application Area Protein Purification " Superdex 75 10/300 GL is used in molecular size exclusion chromatography The total column volume is 24 ml and the maximum proteins in grams to be fractionated should not increased by 50 mg/ml The recommended flow rate is 05 ml/min but for better resolution 02 ml/min is much betterThe void volume peak at 28 minutes in (b) is caused by protein aggregates Column a) HiLoad 16/60 Superdex 75 prep grade b) HiLoad 16/60 Superdex 0 prep grade Sample 1 Myoglobin 15 mg/ml Mw 17 000 2 Ovalbumin 4 mg/ml Mw 43 000 3 Albumin 5 mg/ml Mw 67 000 4 IgG 02 mg/ml Mw 158 000
HiPrep 16/60 Sephacryl S0 HR Void 40 mL IgG 49 Globular proteins Superdex™ 30 prep grade < 1 × 104 Superdex™ 75 prep grade 3 × 1037 × 104 Superdex™ 0 prep grade 1 × 1046 × 105 Dextrans Superdex™ 75 prep grade 5 × 1023 × 104 Superdex™ 0 prep grade 1 × 1031 × 105 Matrix Spherical composite of crosslinkedElution profile of the purified Sumofused DGKαCD from Superdex 75 16/60 column Void volume (V 0) and the elution volume of the standard molecules, γglobulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (13 kDa) are indicated by arrows Most of proteins eluted in a void volume of the column (43 mL)Comparison of the selectivity of Superdex 75 prep grade and Superdex 0 prep grade for model proteins Superdex 75 prep grade (A) gives excellent resolution of the three proteins in the M r range 17 000 to 67 000 while the two largest elute together in the void volume Superdex 0 prep grade (B) resolves these two largest proteins The ferritin
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Superdex pg is supplied in a storage solution of 02 M sodium acetate in % ethanol (Superdex 30 pg and Superdex 75 pg) or in % ethanol (Superdex 0 pg) Ethanol and especially sodium acetate affects the sedimentation prop erties of the resins and so it must be washed off completely before packing the column Superdex 30 pg and Superdex 75 pgGel filtration is performed at 4 °C on a Superdex 75 (26/60) (CABM Rutgers) or Superdex 75 Hiload 16/60 (U of Toronto) equilibrated with the corresponding NMR buffer at a flow rate of 25 ml/min The column is calibrated with LMW gel filtration calibration kitHiLoad 16/60 or 26/60 Superdex 75 prep grade HiLoad 16/60 or 26/60 Superdex 0 prep grade Dismantling tool, Support screens, net ringsμm), Oring, domed nuts Equilibration before a new run Regenerate the column after each run with one column volume of buffer at 30 cm/h (1 ml/min for XK 16/60 or 26 ml/min forXK 26/60)
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Purification And Interactions Of The Muca And Mucb Proteins Constituting The Dna Polymerase Ri Genes And Environment Full Text
Fig 2 Selectivity curves from Superdex 30 pg, Superdex 75 pg and Superdex 0 pg Table 1 Contents of the delivery box Component No supplied Transport device 1 1/16" male connectors 2 Stop plug 1 HiLoad column 1 Instructions 1 Transport device 1/16" male connectors, stopplug Instructions HiLoad 16/600 or 26/600 Superdex 30 pg HiLoad 16Superdex prep grade (pg) is produced by covalent bonding of dextran to highly crosslinked agarose Dextran component determined the separation properties of these media Steep selectivity curves give gppd resolving power for peptides and proteins in the molecular weight range Mr 10 000 600 000 (Superdex 0 pg) For example I have a 14 kDa protein that elutes after 130 ml on a Superdex 75 16/60 (ie after the column volume)!
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(B) SECpurified peptidefree TAPBPR wtH2D b complex was reanalyzed by SEC (Superdex 75) without (solid red trace) and with prior incubation with a 100fold molar excess of highaffinity peptide (ASNENMETM) (dashed red trace) Please note that the extinction coefficient at 280 nm of the MHC I including β2m is 26fold higher than theThe dimer/monomer ratios for the concentrations of 1, 2, and 7 mg/ml are 065, 066, and 080, respectively In the SECFPLC experiment, each protein sample was loaded to a HiLoad 16/60 Superdex 75 prep grade column preequilibrated with the buffer and then eluted at a flow rate of 1 ml/min with a detection of absorbance at 280 nm In this case, all the macromolecules in your sample would elute as one group at the column's void volume The salt or small molecule contaminant that you were seeking to remove would elute very close to the column's total volume Superdex 75 16/60 pg Composite of crosslinked dextran and agarose 34 1 > 370 (NA) Superdex 0
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The void volume of the column (V 0) was 85 ml as determined with blue dextran (not shown) The column volume V c was 212 ml as determined with acetone The elution volumeofmonomericFraCwasV m 279ml,higherthanthe column volume because of the unspecific interaction of the monomeric toxin with the column matrix (16)Pores of the resin and which therefore elute in the void volume In Figure 2, the fraction ranges of globular proteins and peptides with various molecular weights on the newgeneration agarosebased resins can be seen 7 Volume (mL) Molecular weight (M r) 12 17 1 000 000 100 000 10 000 1000 100 10 Superdex 30 Increase Superdex 75 IncreaseBed volume (ml) Recommended sample volume (µl) Theoretical plates (N/m) Recommended flow rate (ml/min) Max flow rate H 2 O at 25° (ml/min) Max pressure over column (MPa) Superdex 0 Increase 10/300 GL 10 x 300 24 25 to 500 48 000 075 18 40 Superdex 0 Increase 5/150 GL 5 x 150 3 4 to 50 42 000 045 075 50 Superdex 0 Increase 32/300
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Hiload 16 60 Superdex 75 Column Ge Healthcare Bioz Ratings For Life Science Research
The AKTAxpress and AKTApurifier FPLC systems, the HisTrap HP column (5 ml, 16 × 25 mm), and HiLoad 16/60 Superdex 0 PG (1 ml, 16 × 600 mm) and HiLoad 16/60 Superdex 75 PG (1 ml, 16 × 600 mm) gel filtration columns were obtained from GEHiLoad Superdex 0 preparation grade resin is well suited for rapid, preparative protein purification and scaleup Its selectivity curve gives a broad fractionation range from M r ~ 10 000 to 600 000, with excellent resolving power for antibody monomers, dimers and contaminants of lower molecular weight, such as albumin and transferrin Read moreAntibody Purification Handbook elution volume is measured from the chromatogram and relates to the molecular size of the molecule V o void volume is the elution volume of molecules that are excluded from the gel filtration medium because they are larger than the largest pores in the matrix and pass straight
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Superdex 75 5/150 GL, Superdex 0 Increase 5/150 GL and of the matrix and therefore elute in the void volume See also Procedure for maintenance and cleaning of SEC columns Sample Acetone mg/ml Sample volume 02% of the total packed column volumeThe void volume of the column was determined with Blue Dextran 00 Prior to each experiment, the column continued with the Superdex 75 (16/60) column having lower molecular mass range State of Aggregation and Glycosylation of IndiVidual Molecules Since the last step in all of the protein purification The MucA' protein (1 ml of 25 mg/ml) was then applied to the gel filtration column Superdex 75 XK 16/60 connected to the FPLC system (Pharmacia, Sweden) equilibrated with mM HEPES pH 74, 10% glycerol w/w, 150 mM NaCl, 01 mM EDTA and 1 mM DTT at a flow rate 08 ml/min The peak of MucA' dimer eluting between 66 and 706 ml was
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Hiload 16 60 Superdex 75 Column Ge Healthcare Bioz Ratings For Life Science Research
I wouldn't worry too much about your apparent MW being 30 kDa and not 40 The( Left ) Overlaid elution profiles from S0 column (the void volume of the column Article Snippet The elution profiles in Figure 3A were obtained from a S0 column (Superdex 0 HiLoad 16/60; Elution profile of the purified Sumofused DGKαCD from Superdex 75 16/60 column Void volume (V 0) and the elution volume of the standard molecules, γglobulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (13 kDa) are indicated by arrows Most of proteins eluted in a void volume of the column (43 mL)
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The hydrodynamic size of the scFv was measured by gel filtration at room temperature (approximately 23 °C) using HiLoad 16/60 Superdex™ 75 and Superdex™ 0 (GE Healthcare) columns, which have a total bed volume (V t) of 1 mL and void volume (V o) of 43 mL The purified scFv was loaded (2 mL injection) onto a column preequilibrated withSuperdex 75 (HR 10/30), and Superdex 0 (HR 16/60) were obtained from Pharmacia The hydroxylapatite HPLC column (BioGel HPHT, 100 x 78 mm) was from BioRad All other chemicals were from Merck (Darmstadt, Federal Republic of Germany (FRG)) or Serva (Heidelberg, FRG)The dimer/monomer ratios for the concentrations of 1, 2, and 7 mg/ml are 065, 066, and 080, respectively In the SECFPLC experiment, each protein sample was loaded to a HiLoad 16/60 Superdex 75 prep grade column preequilibrated with the buffer and then eluted at a flow rate of 1 ml/min with a detection of absorbance at 280 nm
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Size exclusion chromatography using a HiLoad 16/60 Superdex 75 prep grade column on an AKTA FPLC (GE Healthcare, Piscataway, NJ, USA) in 10 mM Tris–HCl pH 74 buffer Pure fractions were checked by SDS polyacrylamide gel electrophoresis (SDSPAGE) and stained with Coomassie blue The extinction coefficient for each protein was calculatedSample volume 1% of the column volume (12 ml for 16/60 or 32 ml for 26/60) Buffer 005 M sodium phosphate, 015 M NaCl, pH 72 or select the buffer in which the sample should be solubilized for the next (25–75 μm) Separation range (M r) globular proteins 1 × 103–1 × 105 (Sephacryl S100 HR)Superdex 0 prep grade for model proteins (A) Superdex 75 prep grade gives excellent resolution of the three proteins in the Mr range 17 000 to 67 000 while the two largest elute together in the void volume (B) Superdex 0 prep grade resolves these two largest proteins The ferritin (5) contains aggregates and thus results in a double peak
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We performed analytical gel filtration with a Superdex 75 16/60 column to determine the molecular mass of PQBP1 by comparing its elution volume (V e) with that of reference proteins The void volume (V 0) was determined with Dextran blue We determined the Stokes radius (R S) of PQBP1 using a linear calibration plot of –log(K av) 1/2Superdex 75 Increase 5/150 GL, Superdex 0 Increase 5/150 GL of the matrix and therefore elute in the void volume The selectivity of a SEC medium depends on its pore size distribution and is described by a selectivity curve (Fig 4) The steeper theGE Healthcare) equilibrated with buffer C (50 mM TrisCl at pH 79, 100 mM NaCl, 50 mM KCl, 5% v/v glycerol) Techniques Sizeexclusion Chromatography
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HiLoad 16/600 and 26/600 Superdex 30 prep grade, HiLoad 16/600 and 26/600 Superdex 75 prep grade, and HiLoad 16/600 and 26/600 Superdex 0 prep grade (pg) are prepacked XK columns designed for 1 One column volume (CV) of low ionic strength buffer at 30 cm/h (1 ml/min for 16/600 or 26 ml/min for 26/600) The Superdex 0 10/300 GL column has a mean bead size 13 um and the fractionation range for the globular proteins is 10,000 – 600,000 The upper exclusion limit for globular proteins that this column can handle is approximately 13x10 6 The bed dimensions for this column are 10 x mm and the column bed volume is approximately 24 ml I am using a Superdex 10/300 column for detecting the interaction of two proteins but I found that both proteins elute in void volume with a small size peak in column volume
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After elution of the desired protein in 1 x SAbuffer with 5 mM 2mercaptoethanol and 10% glycerol we concentrated it using a centrifugal filter unit (MWCO 30 kDa) The concentrated protein was then applied to a ÄKTA purifier equipped with a Superdex 75 16/60HiLoad ® 16/600 Superdex ® 0 prep grade are prepacked XK columns designed for highresolution preparative gel filtration chromatography The column is an excellent choice for separating MAbs from dimers and contaminants Superdex ® prep grade (pg) is produced by covalent bonding of dextran to highly crosslinked agaroseThe separation properties of these Large soluble assemblies (void volume) from a Superdex 75 SEC column did not significantly alter LTP (145 ± 8%, n = 8 vs vehicle alone 158 ± 8%, n = 7, p > 005) (Fig 3b), but when the void volume fraction was incubated at 37 °C for 2 days, the large Aβ assemblies were dissociated into smaller Aβ oligomers that could inhibit hippocampal
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Hiload Acirc quo Cent 16 600 And 26 600 Superdex Acirc quo Cent 30 Prep Grade 16 600
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