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Technical Note: Can Plastic Well Plates Replace Chromatography Autosampler Glass Vials? Product Literature

High coverage gene expression profiling on the Applied Biosystems® 3500xL Genetic Analyzer--a sensitive method for detecting gene transcripts Product Literature

How can I remove salts and detergents from my protein samples before electrophoresis? Product FAQ

Answer

The following protocol can be used to remove salt and detergents from protein samples:
(1) Add 400 µL of methanol per 100 µL of the sample, vortex well.
(2) Add 100 µL of chloroform, vortex.
(3) Add 300 µL distilled water and vortex.
(4) Spin for 1 minute at 14,000 x g.
(5) Remove the top aqueous layer (protein is between the layers).
(6) Add 400 µL of methanol, vortex.
(7) Spin for 2 minutes at 14,000 x g.
(8) Remove as much methanol as possible without disturbing the pellet.
(9) Dry in a vacuum centrifuge (e.g., SpeedVac system ) and add sample buffer for electrophoresis

Reference: Wessel D, Flugge UI (1984) Ana. Biochem 138:141-143.

Answer Id:: E4454

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How can I crosslink antibodies to Dynabeads Protein G magnetic beads? Product FAQ

Answer

Here is a method for crosslinking of IgG immobilized Dynabeads Protein G magnetic beads (5 µg goat a-HSA immobilized to 50 µL Dynabeads Protein G magnetic beads, in PBST), with BS3 linker (Cat. No. 21580):

1. BS3 Stock and Conjugation solutions have to be freshly prepared just before use (i.e., make them after IgG immobilization is complete).
2. Prepare 100 mM BS3 in Conjugation Buffer (Stock Solution). Proceed to making a 5 mM solution by diluting in Conjugation Buffer.
3. Wash the Ig-coupled Dynabeads Protein G magnetic beads twice in 200 µL Conjugation Buffer. Place on magnet and discard supernatant.
4. Resuspend the Dynabeads magnetic beads in 250 µL 5 mM BS3.
5. Incubate for 30 min with tilting/rotation at room temperature.
6. Quench the crosslinking reaction by adding 12.5 µL Quenching Buffer
7. Incubate for 15 min with tilting/rotation at room temperature.
8. Wash crosslinked Dynabeads magnetic beads three times with 200 µL PBST (or IP buffer of your choice), place on magnet, discard supernatant 9. Proceed with your IP and antigen elution

BS3 Conjugation buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7-9.
BS3 Quenching buffer: 1 M Tris HCl, pH 7.5.

Answer Id:: E13021

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How can I cross-link antibodies to Dynabeads magnetic beads? Product FAQ

Answer

Here is a method for cross-linking of IgG immobilized Dynabeads Protein G magnetic beads (5 µg goat a-HSA immobilized to 50 µL Dynabeads Protein G magnetic beads, in PBST), with BS^3 linker from Thermo Scientific (Cat. No. 21580):

(1) BS^3 Stock and Conjugation solutions have to be freshly prepared just before use (i.e., make them after IgG immobilization is complete).
(2) Prepare 100 mM BS^3 in Conjugation Buffer (Stock Solution). Proceed to making a 5 mM solution by diluting in Conjugation Buffer.
(3) Wash the Ig-coupled Dynabeads Protein G magnetic beads twice in 200 µL Conjugation Buffer. Place on magnet and discard supernatant.
(4) Resuspend the Dynabeads magnetic beads in 250 µL 5 mM BS^3.
(5) Incubate for 30 min with tilting/rotation at room temperature.
(6) Quench the cross-linking reaction by adding 12.5 µL Quenching Buffer
(7) Incubate for 15 min with tilting/rotation at room temperature.
(8) Wash cross-linked Dynabeads magnetic beads three times with 200 µL PBST (or IP buffer of your choice), place on magnet, discard supernatant
(9) Proceed with your IP and antigen elution

BS^3 Conjugation buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7-9

BS^3 Quenching buffer: 1 M Tris HCl, pH 7.5.

Answer Id:: E6019

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Flyer: Success with Bioanalysis is a Sum of the Parts Product Literature

How do I anneal my single-stranded DNA oligos to create a ds oligo? Product FAQ

Answer

You will want to anneal equal amounts of the top- and bottom-strand oligos to generate the ds oligos. If your single-stranded oligos are supplied lyophilized, resuspend them in water or TE buffer to a final concentration of 200 µM before use. We generally perform the annealing reaction at a final single-stranded oligo concentration of 50 µM. Annealing at concentrations lower than 50 µM can significantly reduce the efficiency. Note that the annealing step is not 100% efficient; approximately half of the single-stranded oligos remain unannealed even at a concentration of 50 µM. Please see the steps below:

1. In a 0.5 mL sterile microcentrifuge tube, set up the following annealing reaction at room temperature.
“Top-strand” DNA oligo (200 µM) - 5 µL, “Bottom-strand” DNA oligo (200 µM)- 5 µL, 10X Oligo Annealing Buffer - 2 µL, DNase/RNase-Free Water - 8 µL which should make a total volume of 20 µL.
2. If reannealing the lacZ ds control oligo, centrifuge its tube briefly (approximately 5 seconds), then transfer the contents to a separate 0.5 mL sterile microcentrifuge tube.
3. Incubate the reaction at 95 degrees C for 4 minutes.
4. Remove the tube containing the annealing reaction from the water bath or the heat block, and set it on your laboratory bench.
5. Allow the reaction mixture to cool to room temperature for 5-10 minutes. The single-stranded oligos will anneal during this time.
6. Place the sample in a microcentrifuge and centrifuge briefly (approximately 5 seconds). Mix gently.
7. Remove 1 µL of the annealing mixture and dilute the ds oligo as directed.
8. Store the remainder of the 50 µM ds oligo mixture at -20 degrees C.
You can verify the integrity of your annealed ds oligo by agarose gel electrophoresis, if desired.

Answer Id:: E9981

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When expanding my T cells, my cells are dying after activation with Dynabeads magnetic beads? Why is this? Product FAQ

Answer

Here are a few tips to keep the optimal cell culture growth conditions:

- It is important to keep the cell density at 0.5-1.5 x 10E6 cells/mL for optimal growth. When the cell density exceeds 1.5-2.5 x 10E6 cells/mL, re-stimulation is required.
- Re-stimulate every 8-12 days with bead:cell ratio according to the product manual.
- Activate with the correct bead:cell ratio for each specific product according to the manual. Too many beads can inhibit cell activation/proliferation.
- Use optimized cell culture media, add:
-- Growth factors (IL-2, IL-7, IL-15, or other cytokines)
-- Growth enhancing supplements (e.g., human serum/FBS, L-glutamine/glutamax, radical scavenger (10 mM N-acetyl cysteine

Answer Id:: E12077

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What is the binding capacity of Dynabeads Protein G magnetic beads? Product FAQ

Answer

Dynabeads Protein G magnetic beads have a binding capacity of approximately 8 µg human IgG per mg beads. The amount of antibody captured on the beads depends on the affinity of the Protein G to the specific antibody, concentrations of both antibody and Dynabeads Protein G magnetic beads in the starting sample, volumes and binding conditions (pH, salt, etc.) and incubation time. The highest binding capacity noted for these beads under optimal conditions was 640 µg mouse IgG/mL beads; e.g., 100 µL of Dynabeads Protein G magnetic beads will allow isolation of 25 µg mouse IgG1 (about the same affinity as human IgG1, IgG2, IgG3, and IgG4) from a 600 µL sample containing 100 µg per mL mouse IgG1. In this sample, there is a total of 60 µg of mouse IgG1 and immobilization of 25 µg is almost 50%. To ensure the best possible binding in each case, there are a few important factors to consider that influence the binding: the concentration of antibody and beads (keep both high) and the pH of the coupling reaction (which should be kept between 5 and 7). When the total amount of antibody is kept constant but the volume of the reaction is reduced 10 times (from 500 µL to 50 µL), the binding of the antibody is about 30% better. Due to rapid binding kinetics, the maximum amount of Ig binding is obtained after 10-40 minutes of incubation.

Answer Id:: E6009

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Do you have a protocol for purification of His-tagged proteins synthesized in the ExpressWay Cell-Free Expression System? Product FAQ

Answer

Purification may be performed at 4 degrees C or room-temperature depending upon the sensitivity of the synthesized product.

1. Upon completion of incubation, remove the desired portion of reaction for His-tag purification to a clean microcentrifuge tube. Add 4 volumes of Binding buffer and vortex briefly (Add 200 µL for 50 µL of reaction). Centrifuge 5 minutes at 12,000 rpm.
2. Transfer the supernatant to a 2.0 mL tube containing 50 µL pre-equilibrated resin.
3. Incubate with shaking or mixing for 30-60 minutes.
4. Spin down resin for 2 minutes at 800 x g. Do not spin any higher or the resin will collapse and recovery will be low. Carefully remove supernatant.
5. Add 200 µL wash buffer and mix for 5 minutes.
6. Spin down resin for 2 minutes at 800 x g. Carefully remove supernatant.
7. Repeat steps 5 and 6.
8. Add 100 µL Elution Buffer and mix for 5 minutes.
9. Spin down resin for 2 minutes at 800 x g. Carefully remove and save supernatant.
10. Repeat steps 8 and 9.

Binding Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
6 M guanidine HCl (optional)**

Wash Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
15-25 mM imidazole*

Elution Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
150-250 mM imidazole*

**Depending on downstream applications, the purification may be performed under semi-denaturing conditions, or native conditions. Under semi-denaturing conditions, dilute the reaction in denaturing Binding Buffer containing 6 M guanidine HCl; then wash and elute with native buffers.

The concentration of imidazole is dependent upon the type of resin used. For Ni-NTA or ProBond resins, use 25 mM imidazole in the wash buffer and 250 mM imidazole in the elution buffer.

Answer Id:: E12951

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How can I extract peptides from NuPAGE gels for MALDI/MS analysis? Product FAQ

Answer

To extract peptides from NuPAGE gels perform the following protocol:

1) Dehydrate the gel band in 100% methanol for 5 min at room temperature.

2) Rehydrate the gel band in 30% methanol for 5 min.

3) Wash the gel band twice in ultrapure water for 10 min.

4) Wash the gel band three times with 100 mM ammonium bicarbonate containing 30% acetonitrile for 10 min.

5) After the last wash, cut the gel into small pieces and wash the gel pieces in ultrapure water.

6) Dry the gel pieces in a SpeedVac concentrator for 30 min

7) Resuspend the gel pieces in 50 mM ammonium bicarbonate. Add approximately 5 µL buffer per square millimeter of gel. There should be sufficient buffer to cover the gel pieces.

8) Add 5-10 ng/µL trypsin and incubate overnight at 37°C.

9) Centrifuge at maximum speed in a microcentrifuge for 1 min and transfer supernatant to sterile microcentrifuge tube using a clean pipette tip.

10) Extract remaining peptides from the gel with 10-20 µL 50 acetonitrile containing 0.1 trifluoroacetic acid at room temperature. Combine this extract with the supernatant from step 9.

11) Concentrate the sample from step 10 to 4-5 µL using a SpeedVac concentrator and proceed to MALDI/MS analysis. Be sure to include a control sample for MALDI/MS analysis.

Answer Id:: E4145

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How can I remove acetone- and TCA-soluble interferences from my protein samples before electrophoresis? Product FAQ

Answer

The following protocol may be used to remove acetone- and TCA-soluble interferences from protein samples.
(1) Add 2% Na deoxycholate (DOC) to a final concentration of 0.02% of protein solution (for 100 µL sample, add 1 µL 2% DOC).
(2) Mix and store at room temperature for at least 15 min.
(3) Add 100% trichloroacetic acid (TCA) to get a 10% final concentration. Mix and keep at room temperature for at least 1 hour.
(4) Spin at 4 degrees C for 10 min, remove supernatant, and retain the pellet.
(5) Dry tube by inverting it on tissue paper.
(6) Add 200 µL of ice-cold acetone to the TCA pellet. Mix and store on ice for at least 15 min.
(7) Spin for 10 min at 4 degrees C in a microcentrifuge at maximum speed (15000 x g). Remove supernatant as before, air dry the pellet to eliminate any acetone residue.
(8) For electrophoresis, resuspend samples in a minimal volume of sample buffer.
Note: Presence of some TCA can give a yellow color as a consequence of the acidification of the sample buffer; titrate with 1N NaOH or 1M Tris-HCl pH 8.5 to obtain the normal blue sample buffer color.

To prepare 100% TCA: dissolve 1 kg of TCA in 454 mL water. Maintain in a dark bottle at 4 degrees C.

Answer Id:: E4459

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What is your recommended protocol for generating ss phagemid DNA? Product FAQ

Answer

The following protocol can be used to prepare ssDNA from DH12S or DH5aF', DH5aF'IQ, DH11S cells (this strain not currently offered for sale). Use a stock of the helper phage M13KO7 (1) that is of known titer. For convenience, DH12S cells are supplied with M13KO7 helper phage. DH12S cells are both endA positive, so single stranded DNA isolated from these strains tend to be less contaminated with double stranded rf DNA.

Small-Scale Preparation of ss Phagemid DNA:
1. Pick a single colony of cells containing phagemid DNA and resuspend in 2 mL TBG (1.2% tryptone, 2.4% yeast extract 0.4% glycerol, 17 mM KH2PO4 and 55 mM KH2PO4 and 20 mM glucose) containing 100 µg/mL ampicillin in a 15 mL tube.
2. Immediately add 10 µL M13KO7 helper phage stock at 10E11 pfu/mL.
3. Incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 2 hrs.
4. Add kanamycin to a final concentration of 75 µg/mL and incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 18 to 24 hrs.
5. Transfer 1.5 mL of culture to a sterile microcentrifuge tube and pellet cells by centrifuging at 14,000 x g for 10 min at 4 degrees C.
6. Transfer supernatant to fresh microcentrifuge tube and repeat the centrifugation.
7. Transfer 1.2 mL supernatant to a fresh microcentrifuge tube and add 300 µL of 2.5 M NaCl in 40% PEG 4000.
8. Vortex and incubate on ice for 15 min.
9. Centrifuge at 14,000 x g for 15 min at 4 degrees C.
10. Resuspend the pellet in 50 µL TE and phenol extract to remove the viral coat.
11. Use 10 µL of the final 50 µL volume for gel analysis.
This protocol yields 0.5 to 1 µg ss phagemid DNA.

Large-Scale Preparation of ss Phagemid DNA:
1. Resuspend a single colony in 5 mL of TB or LB broth containing 100 µg/mL ampicillin in a 15-mL tube.
2. Shake at 37 degrees C and 275 rpm overnight.
3. Add 100 µL of the overnight culture to 200 mL LB broth and 100 µg/mL ampicillin in a 1 L flask. Incubate at 37 degrees C with shaking (275 rpm) for 3 hrs.
4. Add 200 µL of M13KO7 helper phage (1 x 10E11 pfu/mL) to the culture and continue to incubate for 2 hrs.
5. Add 1.5 mL of 1% (w/v) kanamycin to the cells for a final concentration of 75 µg/mL. Incubate the infected cells for an additional 18 to 24 h at 37 degrees C.
6. Centrifuge this culture at 16,000 x g for 15 min at 4 degrees C .
7. Filter the supernatant through a 0.2 µm sterile filter into an autoclaved centrifuge bottle. Add 40 µL of DNase I (50 units/µL) and incubate at room temperature for 3 hrs. This step should remove any residual ds plasmid DNA contamination.
8. Transfer 100 mL of the supernatant to another centrifuge bottle. Add 25 mL of 2.5 M NaCl in 40% PEG 4000 to each of the centrifuge bottles containing the supernatant.
9. Vortex the mixture, incubate on ice for 1 h, and centrifuge at 16,000 x g for 20 min at 4 degrees C.
10. Carefully discard the supernatant. To fully drain off the remaining solution from the pellets, place the bottles on an angle, with the pellet side facing up for 10 to 15 min. Remove the solution with a sterile Pasteur pipet.
11. Resuspend the pellets in 2 mL of TE buffer. Add 10 µL of proteinase K solution (20 mg/mL), 20 µL of 10% SDS, and incubate this mixture at 45 degrees C for 1 hr.
12. Transfer the digested mixture to three microcentrifuge tubes and extract four times with an equal volume of phenol:chloroform: isoamyl alcohol (25:24:1), precipitate with ethanol, and dissolve in 100 µL TE buffer.
13. Freeze the solubilized DNA at -20 degrees C for 1 hr and centrifuge in a microcentrifuge at 14,000 x g for 15 min at 4 degrees C.
14. Transfer the supernatant containing the ss plasmid DNA to a fresh tube and discard the polysaccharide pellet. Store the ssDNA at 4 degrees C.
15. Determine the DNA concentration (OD260).
This protocol yields ~100 to 200 µg ss phagemid DNA.

(1) Vieira, J. and Messing, J. (1987) Methods in Enzymology 153, 3.

Answer Id:: E4161

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