Vaccine development

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Capturem nickel-functionalized membranes

Purification workflow for Capturem His-Tagged Purification 96-Well Plate. The his-tagged protein of interest is first bound to the membrane, and then washed and eluted with the appropriate buffers. Note: protocol time varies for each format. Check user manuals for specifics on protocol times.

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Capturem spin columns and filtration devices

Available Capturem spin column and filtration device formats. Pictured from left to right: nanoprep, miniprep, 96-well, 24-well, maxiprep, and large-volume Capturem formats. Formats available vary for the different functionalized membranes. Check product pages for list of available formats.

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Load volumes and approximate yields for available Capturem formats by chemistry

Load volumes and yields of Capturem purification formats.

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635714: Capturem His-Tagged Purification 96-Well Plate

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Capturem nickel-functionalized membranes

Purification workflow for Capturem His-Tagged Purification 96-Well Plate. The his-tagged protein of interest is first bound to the membrane, and then washed and eluted with the appropriate buffers. Note: protocol time varies for each format. Check user manuals for specifics on protocol times.

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Capturem spin columns and filtration devices

Available Capturem spin column and filtration device formats. Pictured from left to right: nanoprep, miniprep, 96-well, 24-well, maxiprep, and large-volume Capturem formats. Formats available vary for the different functionalized membranes. Check product pages for list of available formats.

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Load volumes and approximate yields for available Capturem formats by chemistry

Load volumes and yields of Capturem purification formats.

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635724: Capturem His-Tagged Purification Large Volume

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High yield and activity of his-tagged Metridia luciferase purified using the Capturem His-Tagged Purification Maxiprep Kit

High yield and activity of his-tagged Metridia luciferase purified using the Capturem His-Tagged Purification Maxiprep Kit. Panel A. Confirmation of Metridia luciferase expression in 293T cells. The pEF1a-Metluc-6xHis-IRES2-DsRed-Express2 expression vector, which coexpresses 6xhis-tagged Metridia luciferase with a red fluorescent reporter, was constructed using In-Fusion Cloning and the pEF1alpha-IRES-DsRed-Express2 Vector, and transfected into 293T cells using Xfect Transfection Reagent. The DsRed-Express2 reporter was then used to monitor transfection efficiency. Panel B. Yield and activity of Metridia luciferase, pre- and post-purification. Luciferase activity was measured at various steps in the purification of culture supernatant prepared from 293T cells expressing his-tagged Metridia luciferase.

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Capturem his-tagged maxiprep

Capturem his-tagged maxiprep. Capturem his-tagged maxiprep spin columns have a capacity of 2.5 mg his-tagged protein from a starting culture of 25 ml.

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Capturem nickel-functionalized membranes

Purification workflow for Capturem His-Tagged Purification 96-Well Plate. The his-tagged protein of interest is first bound to the membrane, and then washed and eluted with the appropriate buffers. Note: protocol time varies for each format. Check user manuals for specifics on protocol times.

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Capturem spin columns and filtration devices

Available Capturem spin column and filtration device formats. Pictured from left to right: nanoprep, miniprep, 96-well, 24-well, maxiprep, and large-volume Capturem formats. Formats available vary for the different functionalized membranes. Check product pages for list of available formats.

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Load volumes and approximate yields for available Capturem formats by chemistry

Load volumes and yields of Capturem purification formats.

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635713: Capturem His-Tagged Purification Maxiprep Kit

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BCR development.

BCR development. The progenitor cell undergoes recombination of V, D, and J segments in the germline, which generates two identical heavy chains. Recombination of V and J segments generates two identical light chains. Random nucleotide additions or deletions at the junctions of the V, D, and J segments provide additional diversity. Furthermore, B cells activated by immune responses undergo somatic hypermutation (SHM), in which additional point mutations are introduced.

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SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow. Panel A. First-strand cDNA synthesis is dT-primed (BCR dT Primer) and performed by the MMLV-derived SMARTScribe Reverse Transcriptase (RT), which adds nontemplated nucleotides upon reaching the 5' end of each mRNA template. The BCR Oligonucleotide anneals to these nontemplated nucleotides and serves as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT (this is the template-switching step). The BCR Oligonucleotide contains sequence from the Illumina Read Primer 2, serving as a primer-annealing site for subsequent rounds of PCR, and ensuring that only sequences from full-length cDNAs undergo amplification. Panel B. The first PCR uses the first-strand cDNA as a template and includes a forward primer with complementarity to the Illumina Read Primer 2 sequence (BCR Primer 1V), and a reverse primer that is complementary to the constant (i.e., nonvariable) region of BCR heavy or light chains (mBCR Primers 1H, 1K, or 1L). The chains are amplified in separate reactions. By priming from the Read Primer 2 sequence and the constant region, the first PCR specifically amplifies the entire variable region and a considerable portion of the constant region of BCR heavy or light chain cDNA. The second PCR takes the product from the first PCR as a template and uses semi-nested primers (mBCR Primers 2H, 2K, or 2L) to amplify the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. As in PCR 1, the BCR subunit chains are amplified in separate reactions.

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Highly specific amplification of Ig chains.

Highly specific amplification of Ig chains. Libraries containing BCR G and K chain transcripts were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from a hybridoma developed in-house (10E8) and two manufactured ATCC hybridoma samples (HB-8117 and TIB-127). Panel A. Bioanalyzer traces showing gene-specific amplification of G, K and L chains for each hybridoma. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics were determined using MiXCR software (version 2.1.81.8; software not provided with kit) and aligned against all Ig reference sequences, with a clone fraction threshold of 0.01%. V, D, and J IMGT allele outputs, alignment scores and consensus CDR3 amino acid CDR3 for the top heavy (G) and light (K) chain clone for each hybridoma are displayed. For cases in which the MiXCR software determined the presence of more than one V, D, or J allele, all determined alleles with alignment scores are shown.

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PCR cycling and pooling workflow.

PCR cycling and pooling workflow. After RT step, the user amplifies the G, K, or L chain transcripts in separate reactions. Each amplification uses 5 µl of the RT reaction. Following the first PCR, 1 µl of each PCR is used in a separate PCR reaction (PCR 2) to add the same sequencing indexes to each amplified chain for a given sample, but distinct indexes for each different sample. After this final amplification in PCR2, the user may validate each product on a Bioanalyzer or other fragment analysis system. The user then has the flexibility to choose which amplified BCR chains to pool for sequencing.

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Effect of PCR pooling strategy on Ig alignment rate and clonotype identification.

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification. G and K chain PCR products (GK) or G, K and L chain PCR products (GLK) were pooled and sequenced for each hybridoma sample. Panel A. The percentage of reads aligning to Ig reference sequences for GK pooling, GKL pooling, and L only sequencing strategies as determined by MiXCR software. Panel B. The identified CDR3 amino acid consensus sequence and percent distribution for the top heavy (G) and light (K) chain clone for GK pooling or GKL pooling strategies as determined by the MiXCR software. The clone fraction threshold was set to 0.01%.

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634424: SMARTer Mouse BCR IgG H/K/L Profiling Kit

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Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach

Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach. Panel A. Reverse transcription and PCR amplification of TCR subunit mRNA sequences. First-strand cDNA synthesis is primed by the TCR dT Primer and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5′ end of each mRNA molecule, the RT adds non-templated nucleotides to the first-strand cDNA. The SMART-Seq v4 Oligonucleotide contains a sequence that is complementary to the non-templated nucleotides added by the RT, and the oligo hybridizes to the first-strand cDNA. In the template-switching step, the RT uses the remainder of the SMART-Seq v4 Oligonucleotide as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. Full-length variable regions of TCR cDNA are selectively amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (SMART Primer 1) and the constant region(s) of TCR-α and/or TCR-β subunits (TCRa and/or TCRb Mouse Primer 1). A subsequent round of PCR is performed to further amplify variable regions of TCR-α and/or TCR-β subunits and incorporate adapter sequences using a TCR Primer 2 Forward HT Index and a TCRa and/or TCRb Mouse Primer 2 Reverse HT Index. Included in the primers are adapter and index sequences (Read 2 + i7 + P7 and Read 1 + i5 + P5, respectively) that are compatible with the Illumina sequencing platform. Following purification, size selection, and quality analysis, the TCR cDNA library is ready for sequencing. Panel B. Semi-nested PCR approach for amplification of TCR-α and/or TCR-β subunits. The primer pairs used for the first round of amplification capture the entire variable region(s) and most of the constant region(s) of TCR-α and/or TCR-β cDNA. The second round of amplification retains the entire variable region(s) of TCR-α and/or TCR-β cDNA and a smaller portion of the constant region(s). The anticipated size of the final TCR library cDNA (inserts + adapters) is ~700–800 bp.

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Assessment of TCR diversity in mouse spleen samples with the SMARTer Mouse TCR a/b Profiling Kit

Assessment of TCR diversity in mouse spleen samples with the SMARTer Mouse TCR a/b Profiling Kit. Panel A. To compare TCR diversity between the spleen's general T-cell population and purified CD4+ T cells, total splenocytes were isolated from spleen and divided into two groups. The first group was left untouched, and the second group was subject to CD4+ T-cell purification (see Methods section for details). Total RNA was extracted from the total splenocyte group and the group enriched for CD4+ T cells, and each RNA sample was used for library preparation. Panel B. Proportion of on-target reads mapping to TCR-α or TCR-β CDR3 sequences. Panel C. Rarefaction curves showing the relationship between read depth and the number of unique clonotypes identified for TCR-α (left) and TCR-β (right). When a curve starts to flatten off, the number of unique clonotypes identified is reaching saturation. Dashed lines show the theoretical number of clonotypes as the number of reads increases.

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Assessment of TCR-α clonotype diversity in various tissues of transgenic mice of different ages with the SMARTer Mouse TCR a/b Profiling Kit

Assessment of TCR-α clonotype diversity in various tissues of transgenic mice of different ages with the SMARTer Mouse TCR a/b Profiling Kit. Panel A. The percentage of reads mapping to TCR-α CDR3 sequences identified in the thymus, spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and large intestine (LI) is consistent across the 16-week-old, 34-week-old, and 38-week-old mice. Panel B. The immune tissues showed the highest TCR-α clonotypic diversity, while the large intestine showed the lowest diversity. This indicates that the large intestine has the lowest T-cell infiltrate. Panel C. Abundance of the TRAV3-2-TRAJ58 clonotype, expressed as a percentage of all reads mapping to any TCR-α or TCR-β clonotype, across tissue types and ages. Panel D. Abundance of the TRAV16N-TRAJ56 clonotype, expressed as a percentage of all reads mapping to any TCR-α or TCR-β clonotype, across tissue types in the 38-week-old mouse.

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634404: SMARTer Mouse TCR a/b Profiling Kit

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Tunable selection of DNA fragments in a given size range using NucleoMag NGS Clean-up and Size Select

Tunable selection of DNA fragments in a given size range using NucleoMag NGS Clean-up and Size Select. NucleoMag NGS Clean-up and Size Select can be used to isolate DNA fragments of a given size within a target range of ~150�800 bp. Selective binding of DNA fragments above a designated size cutoff is achieved by applying NucleoMag beads to the sample at a predetermined dilution ratio. This approach can be applied once or twice to perform single- or double-sided size selection. In the latter case, an initial round of bead purification is typically performed to bind and remove fragments above the upper size cutoff, and a second round of purification is performed to bind and retain fragments above the lower size cutoff. Dilution ratios used for size selection with NucleoMag beads are similar to ratios employed by size-selection beads from other providers, such that NucleoMag beads can be incorporated in existing NGS library preparation workflows without protocol adjustment.

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Relationship between bead-to-sample volume ratio and recovery rate for DNA fragments of different sizes with NucleoMag NGS Clean-up and Size Select

Relationship between bead-to-sample volume ratio and recovery rate for DNA fragments of different sizes with NucleoMag NGS Clean-up and Size Select. Varying quantities of NucleoMag beads were applied to 100-?l aliquots containing mixtures of DNA fragments ranging in size from 100 bp to 1,000 bp (10 ng/?l) in the indicated dilution ratios (gray row). Recovery rates for fragments of each size are expressed as a percentages (%).

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Examples of double-sided size selection using NucleoMag NGS Clean-up and Size Select

Examples of double-sided size selection using NucleoMag NGS Clean-up and Size Select. Sheared mouse genomic DNA was size selected with NucleoMag beads using two different pairs of dilution ratios and measured using a Bioanalyzer. Green trace: sheared mouse genomic DNA prior to size selection. Red trace: size distribution after double-sided size selection using dilution ratios of 0.4 (right) and 0.6 (left), resulting in a mean fragment size of 460 bp. Blue trace: size distribution after double-sided size selection using dilution ratios of 0.55 (right) and 0.8 (left), resulting in a mean fragment size of 340 bp.

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Fragment size analysis for NGS libraries prepared using size-selection beads from various providers

Fragment size analysis for NGS libraries prepared using size-selection beads from various providers. NGS libraries were prepared from sheared E. coli DNA using the TruSeq DNA PCR-Free kit by Illumina and size selected using magnetic beads from different providers (Illumina, AMPure XP, and NucleoMag NGS Clean-up and Size Select). Panel A. Bioanalyzer trace indicating size distribution of sheared E. coli DNA (1-?g input) prior to NGS library preparation. Panel B. Bioanalyzer traces indicating size distributions of resulting NGS libraries purified using Illumina (red), AMPure XP (blue), or NucleoMag (green) magnetic beads.

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Comparison of sequencing data generated from NGS libraries prepared using size-selection beads from various providers

NGS libraries were prepared from sheared E. coli DNA using the TruSeq DNA PCR-Free kit by Illumina and size selected using magnetic beads from different providers (Illumina, AMPure XP, and NucleoMag NGS Clean-up and Size Select). Panel A. Bioanalyzer trace indicating size distribution of sheared E. coli DNA (1-?g input) prior to NGS library preparation. Panel B. Bioanalyzer traces indicating size distributions of resulting NGS libraries purified using Illumina (red), AMPure XP (blue), or NucleoMag (green) magnetic beads.

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744970.50: NucleoMag NGS Clean-up and Size Select

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The NucleoSpin 8/96 Procedure

The NucleoSpin 8/96 Procedure.

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Reproducible yields of plasmid DNA are obtained with the NucleoSpin 96 Plasmid kit

Reproducible yields of plasmid DNA are obtained with the NucleoSpin 96 Plasmid kit. The kit was used to purify pBluescript from E. coli DH5 alpha grown in 1.3 ml LB medium (upper row) or 2xYT medium (lower row), and 15 µl samples of each prep were analyzed on a 1% TAE agarose gel. Size maker: lambda HindIII.

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Consistent yields of plasmid DNA are obtained with the NucleoSpin 8 Plasmid kit

Consistent yields of plasmid DNA are obtained with the NucleoSpin 8 Plasmid kit. The kit was used to prepare 16 preps of pUC18 from E. coli DH5 alpha, and a sample of each prep was analyzed on a 1% TAE agarose gel. Size marker: 1 kb ladder.

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740625.4: NucleoSpin 96 Plasmid

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NucleoBond Xtra Columns: The enlarged columns lead to lower silica resin beds and enable faster flow of lysate and buffers through the columns, allowing for fast purification

NucleoBond Xtra Columns: The enlarged columns lead to lower silica resin beds and enable faster flow of lysate and buffers through the columns, allowing for fast purification. The improved silica material is based on the anion-exchange group MAE.

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The NucleoBond Xtra Combi Rack is specifically designed to support both Midi and Maxi columns

The NucleoBond Xtra Combi Rack is specifically designed to support both Midi and Maxi columns. Combi Racks facilitate column handling, loading, and elution.

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The bacterial lysate is cleared and loaded onto the equilibrated column in one step (plasmid DNA binds to the anion exchange resin)

The bacterial lysate is cleared and loaded onto the equilibrated column in one step (plasmid DNA binds to the anion exchange resin). The first washing step using equilibration buffer is performed with the column filter inserted into the column to wash out residual lysate from the filter and obtain maximum recovery of DNA. After subsequent washing, the purified plasmid DNA is eluted in a high-salt buffer and precipitated with isopropanol. Precipitation can be performed by centrifugation [NucleoBond Xtra Midi (midiprep) or Maxi (maxiprep) kits] or by using the NucleoBond Finalizer.

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740414.50: NucleoBond Xtra Maxi

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Higher yields versus Qiagen with NucleoBond Xtra Midi and NucleoBond Xtra Maxi

Higher yields than Qiagen with NucleoBond Xtra Midi and NucleoBond Xtra Maxi. Plasmid DNA was purified from bacterial cultures using NucleoBond Xtra Midi, NucleoBond Xtra Maxi, and corresponding midiprep and maxiprep kits from Qiagen following the protocols for each kit (in each case, the maximum input volume specified by the corresponding kit protocol was processed). Yields of plasmid DNA were 60–100% higher with the NucleoBond kits than the Qiagen kits (Q Midi = Qiagen Plasmid Midi Kit, Q Maxi = Qiagen Plasmid Maxi Kit).

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NucleoBond Xtra Columns: The enlarged columns lead to lower silica resin beds and enable faster flow of lysate and buffers through the columns, allowing for fast purification

NucleoBond Xtra Columns: The enlarged columns lead to lower silica resin beds and enable faster flow of lysate and buffers through the columns, allowing for fast purification. The improved silica material is based on the anion-exchange group MAE.

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The NucleoBond Xtra Combi Rack is specifically designed to support both Midi and Maxi columns

The NucleoBond Xtra Combi Rack is specifically designed to support both Midi and Maxi columns. Combi Racks facilitate column handling, loading, and elution.

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The bacterial lysate is cleared and loaded onto the equilibrated column in one step (plasmid DNA binds to the anion exchange resin)

The bacterial lysate is cleared and loaded onto the equilibrated column in one step (plasmid DNA binds to the anion exchange resin). The first washing step using equilibration buffer is performed with the column filter inserted into the column to wash out residual lysate from the filter and obtain maximum recovery of DNA. After subsequent washing, the purified plasmid DNA is eluted in a high-salt buffer and precipitated with isopropanol. Precipitation can be performed by centrifugation [NucleoBond Xtra Midi (midiprep) or Maxi (maxiprep) kits] or by using the NucleoBond Finalizer.

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740424.50: NucleoBond Xtra Maxi EF

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740491.4: NucleoSpin 96 Plasmid Transfection-grade

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Nickel resin—His60 for his-tag purification of proteins

Nickel resin—His60 for his-tag purification of proteins.

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635661: His60 Ni Superflow Resin

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SDS-PAGE of TALON CellThru Resin purified proteins

SDS-PAGE of TALON CellThru Resin purified proteins. E. coli BL21 cells were sonicated in TALON wash buffer and run through a TALON CellThru column eluted in 150 mM imidazole. Note that some target protein is trapped in membrane fractions and does not get absorbed on the column. M: molecular weight marker.

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His-tag purification TALON metal affinity resins are available in different sizes

His-tag purification TALON metal affinity resins are available in different sizes.

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Native vs. denaturing purification procedures using TALON resin

Native vs. denaturing purification procedures using TALON resin.

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Native purification with TALON resin preserves biological activity of proteins

Native purification with TALON resin preserves biological activity of proteins. Fresh cells (0.5 g) expressing 6xHis-GFPuv were extracted in 5 ml of 50 mM sodium phosphate; 0.3 M NaCl, pH 7.0 Panel A. Elution profile of GFP which was loaded, washed with the same buffer, and eluted with a step gradient of imidazole (150 mM). Panel B. Fractions were analyzed by SDS-PAGE. The fluorescent signal of green fluorescent protein (GFPuv) was completely enriched by TALON Superflow Resin.

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Purification of 6xHis-GFPuv under denaturing conditions using TALON resin

Purification of 6xHis-GFPuv under denaturing conditions using TALON resin. The fusion protein was purified in 8 M urea using TALON resin. M=molecular weight markers.

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Native purification of 6xHis protein using TALON resin in the presence of beta-mercaptoethanol

Native purification of 6xHis protein using TALON resin in the presence of beta-mercaptoethanol. N-terminal 6xHis-tagged mouse DHFR (19.5 kDa) was expressed in E. coli. 2 ml of lysate was purified using gravity flow on TALON resin in increasing concentrations of beta-mercaptoethanol. Even lanes: 20 μl of nonadsorbed material. Odd lanes: 5 μl of eluate

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Protein purification yields in the presence of beta-mercaptoethanol with TALON resin compared to Ni-NTA resin

Protein purification yields in the presence of beta-mercaptoethanol with TALON resin compared to Ni-NTA resin. N-terminal 6xHis DHFR was expressed and purified under native conditions. Protein concentrations were determined by Bradford assay. Yields are expressed as a percentage of total protein in the cell lysate.

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635502: TALON Metal Affinity Resin

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Sensitive and reproducible clonotype detection from a broad range of sample types and RNA amounts.

Variable and highly complex samples are accommodated without the need for RNA purification or T-cell enrichment. TRA and TRB libraries were generated from 1, 10, and 100 ng of human CD3+ T‑cell total RNA (Panel A), 1,000 and 10,000 CD3+ T cells (Panel B), and 20 ng of whole-blood RNA extracted from three different samples (Panel C). The sequence reads were processed by Cogent NGS Immune Profiler.

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The Takara Bio TCR profiling kit identifies a wide range of clonotype counts.

The number of clonotypes detected reveals biological variation. Duplicate libraries were generated from 10 ng RNA extracted from single-donor PBMC samples (P1-P6) and sequenced on an Illumina MiSeq system using 300-bp paired-end reads to obtain approximately 1.5 million reads per sample. Resulting sequencing reads were processed with Cogent NGS Immune Profiler. Clonotype numbers from different donors for TRA (blue) and TRB (orange) are shown. Error bars shown represent the standard error between the duplicates.

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The Takara Bio TCR profiling kit generates data with sensitivity superior to competitors

A dramatically higher clonotype number was observed for TRB after downsampling. (TRA results were similar; data not shown.) 5M PBMC cells were split from two different healthy donors for RNA and gDNA extraction. 1.6 g of gDNA (15% of the total amount of extracted gDNA) was used for library preparation according to manufacturer's instructions. 100 ng of RNA (2% of the total amount of extracted RNA) was used for library preparation.

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634478: SMARTer Human TCR a/b Profiling Kit v2

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Sensitive and reproducible clonotype detection from a broad range of sample types and RNA amounts.

Variable and highly complex samples are accommodated without the need for RNA purification or T-cell enrichment. TRA and TRB libraries were generated from 1, 10, and 100 ng of human CD3+ T‑cell total RNA (Panel A), 1,000 and 10,000 CD3+ T cells (Panel B), and 20 ng of whole-blood RNA extracted from three different samples (Panel C). The sequence reads were processed by Cogent NGS Immune Profiler.

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The Takara Bio TCR profiling kit identifies a wide range of clonotype counts.

The number of clonotypes detected reveals biological variation. Duplicate libraries were generated from 10 ng RNA extracted from single-donor PBMC samples (P1-P6) and sequenced on an Illumina MiSeq system using 300-bp paired-end reads to obtain approximately 1.5 million reads per sample. Resulting sequencing reads were processed with Cogent NGS Immune Profiler. Clonotype numbers from different donors for TRA (blue) and TRB (orange) are shown. Error bars shown represent the standard error between the duplicates.

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The Takara Bio TCR profiling kit generates data with sensitivity superior to competitors

A dramatically higher clonotype number was observed for TRB after downsampling. (TRA results were similar; data not shown.) 5M PBMC cells were split from two different healthy donors for RNA and gDNA extraction. 1.6 g of gDNA (15% of the total amount of extracted gDNA) was used for library preparation according to manufacturer's instructions. 100 ng of RNA (2% of the total amount of extracted RNA) was used for library preparation.

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634479: SMARTer Human TCR a/b Profiling Kit v2

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The In-Fusion cloning protocol

The In-Fusion cloning protocol.

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638956: In-Fusion Snap Assembly EcoDry Master Mix