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FAQ - His Tag Protein Purification

  1. Is it necessary to regenerate the column each time?
  2. The manual mentions that the Lysis Equilibration Buffer, Wash Buffer and Elution buffer contain 50 mM NaH2PO4 and 300 mM NaCl. Is it possible to use 50mM PBS instead of them?
  3. What concentration of PMSF is needed for cell lysis and protein purification?

FAQ - His Tag Protein Purification

Answers:

  1. Is it necessary to regenerate the column each time?

    No. You can regenerate the column every 3-5 times.

  2. The manual mentions that the Lysis Equilibration Buffer, Wash Buffer and Elution buffer contain 50 mM NaH2PO4 and 300 mM NaCl. Is it possible to use 50mM PBS instead of them?

    The concentration of PBS is lower. It will result in some non-specific binding.

  3. What concentration of PMSF is needed for cell lysis and protein purification?

    We recommend a final concentration of PMSF at 1mM.

Trouble shooting - His Tag Protein Purification

Problems

Possible Cause

Suggestion

No binding
The column is not equilibrated sufficiently in the buffer.
Repeat or try a longer equilibration step.
Protein precipitation in the column filter
Clean the column, exchange or clean the filter
Purification buffers contain chelating agents such as EDTA or EGTA.
Prepare new buffer without chelating agents
Histidine tag is buried within the protein and unavailable to bind under native conditions.
Use another fusion tag, or try fusing the histidine tag to the other end of the protein
Low yield
The concentration of imidazole in purification buffer and washing buffer is high. Binding affinity to the resin is weak.
Optimize imidazole concentration to be specific to your protein
Degraded by proteases
Add protease inhibitors.
Protein is still attached to ligand.
Try a higher concentration of imidazole
High flow rates may not allow sufficient time for the proteins to bind and can be lost in the flow through.
Flow rate is critical to protein binding. Use lower flow rate, or try batch binding.
Overloading the column can reduce the protein yield
The recommended rule is  to keep the protein load  within 50–75% of the column volume.
Purity
Host proteins contain multiple tandem histidine residues, they can bind weakly to the nickel resin and co-elute with the protein of interest.
Low concentration of imidazole can be added to the lysis and wash buffers to elute the nonspecific binding protein.
Host chaperone protein (HCP) is a common contaminant
MgCl2 and ATP can be used to remove the chaperone proteins

Trouble shooting - GST Tag Protein Purification

Problem
Probable Cause
Suggestion
The yield of the purified GST fusion protein is low or undetectable.
The fusion protein forms inclusion bodies.
Grow bacteria at a lower temperature (20-30°C), or reduce final concentration of IPTG to 0.1 mM for protein induction, or reduce the induction time.
Properly dissolve and refold the inclusion body prior to the purification.
The fusion protein does not bind to Glutathione Resin efficiently.
Use batch method for purification.  Incubate clear solution (then sonicate, etc.) containing GST-fusion protein with Glutathione Resin for 2 hours or longer (i.e. overnight) and then load the mixture onto the column.
The fusion protein does not contain active GST.
Use mild sonication condition or other lysis method, such as lysozyme so that GST is not denatured.
The fusion protein is degraded by protease.
Add appropriate protease inhibitors such as PMSF in the lysis solution and wash solution.
The fusion protein is not efficiently eluted from Glutathione Resin.
Increase elution time or increase the concentration of glutathione to 15 mM or higher in the elution buffer.
Adjust the pH of the elution buffer to 8.0-9.0 without increasing the glutathione concentration.
Add Triton X-100 (0.1%, final concentration) or n-Octylglucoside (2%, final concentration) or NaCl (0.1-0.2 M, final concentration) to the elution buffer.
Multiple bands observed in the eluted protein
The fusion protein is degraded by protease.
Add appropriate protease inhibitors (or inhibitor cocktails) such as PMSF in the lysis solution and wash solution.
Some host proteins, such as chaperonins, may interact with the fusion protein.
Add DTT (5 mM, final concentration) in the wash buffer.  Incubate the recombinant protein solution in chaperonin buffer (2 mM ATP, 10 mM MgSO4, 50 mM Tris-HCl) at 37°C for 10 min prior to the purification.
Over-sonication will cause some protein to bind to the fusion protein.
Use milder sonication condition or another lysis method.
Some protein will bind to the fusion protein or beads non-specifically.
Optimize the wash conditions.  Detergents such as 1% Triton X-100, 1% Tween-20, 0.03% SDS, or 0.1% NP-40 may be used to reduce non-specific binding.  Salt concentration in the wash solution can also be optimized to reduce non-specific binding.

Trouble shooting – Antibody Purification

Problem
Possible Cause
Suggestion
The flow rate of the column is very low (<0.5 ml/minute).
Tiny air bubbles from buffer or particles from sample block the gel pores.
De-gas buffers and samples.  Do not allow the column to dry.
A considerable amount of sample has been loaded, but no specific antibody of interest is detected.
The concentration of the antibody of interest is very low.
Purify the antibody using the specific antigen coupled to a resin (i.e., High-Affinity Iodoacetyl Resin, Cat. No. L00403).
The antibody is degraded.
The antibody is sensitive to low-pH
elution buffer
Neutralize the eluted fractions with Neutralization Buffer immediately after elution.
No antibody is detected in any elution fraction.
The IgG subclass does not bind to this resin.
Try other affinity chromatography media to purify the antibody, such as Protein G Resin or Protein L Resin.

Trouble shooting - Immunoprecipitation

Problems
Possible Cause
Suggestion
High background
Incomplete washing
Increase the number of washing steps and prolong the washing steps. Background can be avoided using GenScript MagBeads.
Protein degradation during IP
Add fresh-prepared protease inhibitors when sample is lysed.
Non-specific binding
The structure of agarose beads will absorb non-specific protein.
Pre-incubate agarose beads with the lysate before immunoprecipitation. Using  GenScript's MagBeads will eliminate this step.
No target protein detected
Expression level of target protein is low in the sample.
Increase the amount of lysate  

FAQ - eStain® 2.0 Protein Staining System

  1. What is the difference between eStain® 2.0 Protein Staining System and original eStain® System?
  2. Why should I use the eStain® 2.0 Protein Staining System?
  3. What's the difference between the eStain® 2.0 Protein Staining System and conventional Coomassie blue staining methods?
  4. What are the functions of eStain® Protein Staining Pads?
  5. What is the difference between the eStain® Protein Staining Pads R-250 and G-250?
  6. Can the eStain® Protein Staining Pads be re-used for staining multiple gels?
  7. Can the eStain® Protein Staining Pads be used on other blotting devices to stain protein gels?
  8. Why should I replace the eStain® 2.0 Graphite Electrode?
  9. Which types of protein gels are eStain® 2.0 Protein Staining System compatible with?
  10. How many gels can be stained at the same time?
  11. What is the sensitivity of the eStain® 2.0 Protein Staining System?
  12. Is the eStain® 2.0 Protein Staining System compatible with mass spectrometry and 2D electrophoresis?
  13. Can a Western blot be performed on a gel that has been stained with eStain® 2.0 Protein Staining System?
  14. Can a Western blot be performed on a gel that has been stained with eStain® 2.0 Protein Staining System?
  15. What is the recommended temperature when using the eStain® 2.0 Protein Staining System?
  16. Why is there a small crack at the titanium cathode side of the lid of the eStain® 2.0 Device after a period of use?
  17. Can I start a run without the assembled staining stack (eStain Anode Pad-Gel-eStain Cathode Pad) in place?

FAQ - eBlot™ Protein Transfer System

  1. What's the difference between the eBlot™ Protein Transfer System and conventional blotting methods?
  2. Which types of protein gels are compatible with the eBlot™ Protein Transfer System?
  3. How many gels can be blotted at the same time?
  4. What are the functions of eBlot™ Protein Transfer Pads?
  5. Which eBlot™ Protein Transfer Pads should I choose?
  6. Can the eBlot™ Protein Transfer Pads be re-used for blotting multiple gels?
  7. Why should I replace the eBlot™ Graphite Electrode and how often should I do it?
  8. Can I blot very thin (less than 1 mm) or very thick (more than 1 mm) gels using the eBlot™ Protein Transfer System?
  9. What is the recommended temperature for using eBlot™ Protein Transfer System?
  10. Why is there a small crack at the titanium cathode side of the lid of the eBlot™ Protein Transfer device after a period of use?
  11. Can I start a run without the assembled transfer stack (eBlot™ Anode Pad-Membrane-Gel-Gel Window-eBlot™ Protein Transfer Cathode Pad) in place?

FAQ - eStain® 2.0 Protein Staining System

Answers:

  1. What is the difference between eStain® 2.0 Protein Staining System and original eStain® System?
  2. eStain® 2.0 Protein Staining System is an upgrade of the original eStain® 1.0 System. The eStain® 2.0 Protein Staining Device is a totally redesigned electric staining unit. The stylish and durable design delivers a more user-friendly unit with extended service life. A larger LCD screen on the control panel clearly displays all the parameters. A waste tray is added to the right side of eStain® 2.0 Device to collect liquid waste and simplify the cleanup. The optimized circuit and control program increases reliability and reproducibility. The eStain® 2.0 Device uses a new eStain® 2.0 Graphite Electrode (L02017) which cannot be used on the original eStain®1.0 Device. The eStain® Protein Staining Pads (R-250, L02011 and G250, L02012) are compatible with both eStain® 2.0 System and the original eStain® 1.0 System.

  3. Why should I use the eStain® 2.0 Protein Staining System?
  4. The eStain® 2.0 Protein Staining System is an innovation in protein electrophoresis analysis. It is designed to quickly, easily and reliably perform protein gel staining with Coomassie blue dye within 7 minutes or less. This system can greatly shorten your workflow and save valuable research time for subsequent analysis.

  5. What's the difference between the eStain® 2.0 Protein Staining System and conventional Coomassie blue staining methods?
  6. Conventional Coomassie blue staining methods rely on Brownian motion and require three steps including fixing (or washing), staining and de-staining (or washing), etc., which is a long and tedious process. The eStain® 2.0 Protein Staining System applies GenScript's proprietary electric staining technology. A definitive voltage is applied to the graphite anode and titanium cathode to drive the negatively charged Coomassie blue dye into the gel matrix to stain the proteins and to push the unbound Coomassie blue dye out of the gel matrix to destain the rest of the gel. The eStain® 2.0 System integrates three steps of traditional methods into a single step and significantly reduces the time required for protein staining analysis.

  7. What are the functions of eStain® Protein Staining Pads?
  8. The eStain® Protein Staining Pads are the consumable part of the eStain® 2.0 System. Each pack of eStain® Protein Staining Pads contains an eStain® Cathode Pad presoaked with proprietary cathode buffer with CBB dye R-250 or G-250 incorporated, and an eStain® Anode Pad presoaked with proprietary anode buffer. Similar to semi-dry blotting, the eStain® Cathode Pad and eStain® Anode Pad act as ion reservoirs. The eStain® Cathode Pad also supplies negatively charged CBB dye for protein staining.

  9. What is the difference between the eStain® Protein Staining Pads R-250 and G-250?
  10. The eStain® Pads (R-250) have incorporated Coomassie blue dye R-250 and generate dark blue protein bands. The eStain® Pads (G-250) have incorporated Coomassie blue dye G-250 and generate bright blue protein bands. Customers can select R-250 or G-250 eStain® Pads according to their color preference.

  11. Can the eStain® Protein Staining Pads be re-used for staining multiple gels?
  12. No. For best results, one pack of eStain® Protein Staining Pad is used to stain one gel. Discard used eStain® Pad after each staining.

  13. Can the eStain® Protein Staining Pads be used on other blotting devices to stain protein gels?
  14. No. The eStain® Protein Staining Pads are designed to work with the eStain® 1.0 and eStain® 2.0 Protein Staining Devices. If they are used on other blotting devices, we cannot guarantee users satisfactory staining results.

  15. Why should I replace the eStain® 2.0 Graphite Electrode?
  16. During the electric staining process, the eStain® 2.0 Graphite Electrode will absorb ions from the anode pad while losing carbon composition, which will change the conductivity of the graphite electrode, thereby affecting the staining results. For best staining results, after 100 uses, the worn eStain® 2.0 Graphite Electrode should be replaced by a new one; the graphite electrode should also be replaced if staining performance is significantly reduced prior to 100 uses.

  17. Which types of protein gels are eStain® 2.0 Protein Staining System compatible with?
  18. The eStain® 2.0 Protein Staining System is designed to work with multiple homemade or precast mini polycrylamide gels, including Tris-Glycine, Bis-Tris, Tris-Acetate and Tris-Tricine gels, etc. For specially formulated gels, optimization of the staining time may be needed for best results.

  19. How many gels can be stained at the same time?
  20. Using eStain® 2.0 Protein Staining System, one gel can be stained for each run.

  21. What is the sensitivity of the eStain® 2.0 Protein Staining System?
  22. The eStain® 2.0 System uses Coomassie blue dye R-250 and G-250 as staining reagents. They have a similar detection sensitivity down to a few dozen nanograms per protein band.

  23. Is the eStain® 2.0 Protein Staining System compatible with mass spectrometry and 2D electrophoresis?
  24. Yes. It is compatible with mass spectrometry and 2D electrophoresis just like conventional Coommassie blue staining methods.

  25. Can a Western blot be performed on a gel that has been stained with eStain® 2.0 Protein Staining System?
  26. No. To ensure optimal Western blot results, gels without staining (either conventional Coomassie blue staining or eStaining) should be used.

  27. Can a Western blot be performed on a gel that has been stained with eStain® 2.0 Protein Staining System?
  28. Yes. However, the staining time needs to be optimized. For example, 6 -7 minutes are needed to stain a 0.75 mm mini gel and 8-9 minutes are needed to stain a 1.5 mm mini gel.

  29. What is the recommended temperature when using the eStain® 2.0 Protein Staining System?
  30. For best staining results, it is recommended to use eStain® 2.0 Protein Staining System at room temperature (22 - 28 ℃). If temperature is below 22 ℃, users may need to extend the running time to obtain satisfied staining results based on gel thickness.

  31. Why is there a small crack at the titanium cathode side of the lid of the eStain® 2.0 Device after a period of use?
  32. A tiny crack may develop at one of the two lower corners next to the titanium cathode due to a thermal expansion difference between the lid material and the titanium cathode. However, it does not affect the staining performance of the device.

  33. Can I start a run without the assembled staining stack (eStain Anode Pad-Gel-eStain Cathode Pad) in place?
  34. No. It will DAMAGE the device due to a short circuit. We have placed a warning sign on the lid of the eStain® and on the cover page of the User Manual.

FAQ - eBlot™ Protein Transfer System

Answers:

  1. What's the difference between the eBlot™ Protein Transfer System and conventional blotting methods?
  2. Conventional wet and semi dry blotting processes take hours to complete and results can be unpredictable. The eBlot™ Protein Transfer System applies GenScript's proprietary electric transfer technology, where a definitive voltage is applied to the graphite anode and titanium cathode to drive negatively charged proteins from a mini polyacrylamide gel onto the membrane. The eBlot™ Protein Transfer System significantly reduces the time required for protein transfer, from hours down to 7 - 10 minutes.

  3. Which types of protein gels are compatible with the eBlot™ Protein Transfer System?
  4. The eBlot™ Protein Transfer System is designed to work with multiple homemade or precast mini polycrylamide gels, including Tris-Glycine, Bis-Tris, Tris-Acetate and Tris-Tricine gels, etc. For specially formulated gels, optimization of the transfer time may be needed for best results.

  5. How many gels can be blotted at the same time?
  6. One mini gel can be blotted per run.

  7. What are the functions of eBlot™ Protein Transfer Pads?
  8. The eBlot™ Protein Transfer Pads are the consumable part of eBlot™ Protein Transfer System. Each pack of eBlot™ Protein Transfer Pad contains an eBlot™ Cathode Pad (white) and Anode Pad (yellow) presoaked with proprietary buffers, acting as ion reservoirs.

  9. Which eBlot™ Protein Transfer Pads should I choose?
  10. We offer transfer pads bundled with membrane: Nitrocellulose (cat. no. L03013) or PVDF (cat. no.L03014). If you already have your own membrane, you can choose our basic package without membrane (cat. no. L03011).

  11. Can the eBlot™ Protein Transfer Pads be re-used for blotting multiple gels?
  12. No. For best results, one pack of eBlot™ Protein Transfer Pad is used for one gel. Discard used eBlot™ Protein Transfer Pads after each blotting.

  13. Why should I replace the eBlot™ Graphite Electrode and how often should I do it?
  14. During the electric transfer process, the eBlot™ Graphite Electrode will absorb ions from the anode pad while losing carbon composition, which will change the conductivity of the graphite electrode, thereby affecting the blotting results. For best results, after 100 uses, the worn eBlot™ Graphite Electrode should be replaced with a new one. The graphite electrode should also be replaced if blotting performance is significantly reduced prior to 100 uses.

  15. Can I blot very thin (less than 1 mm) or very thick (more than 1 mm) gels using the eBlot™ Protein Transfer System?
  16. Yes. However, the transfer time needs to be optimized. Also, for 0.75-1mm thickness gels, use 2 pieces of sponge cushion underneath the electrode; for 1.5 mm gels, use only 1 piece of sponge cushion.

  17. What is the recommended temperature for using eBlot™ Protein Transfer System?
  18. For best staining results, it is recommended to use the system at room temperature (22-28 ℃). If temperature is below 22 ℃, users may need to extend the running time to obtain satisfied results based on gel thickness.

  19. Why is there a small crack at the titanium cathode side of the lid of the eBlot™ Protein Transfer device after a period of use?
  20. A tiny crack may develop at one of the two lower corners next to the titanium cathode due to thermal expansion difference between the lid material and the titanium cathode. However, it does not affect the transfer performance of the device.

  21. Can I start a run without the assembled transfer stack (eBlot™ Anode Pad-Membrane-Gel-Gel Window-eBlot™ Protein Transfer Cathode Pad) in place?
  22. No. It will DAMAGE the device due to short circuit.

 
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