What methods are available to elute the AviTag’d protein off the AbCA?
Elution methods include low pH elution, polyol elution with propanediol or ethylene glycol with a non-chaotropic salt, or we have even had a customer elute their AviTag’d protein off the AbCA by competing it off with synthesized AviTag™ peptide in solution.AbCA
Does the AbCA resin come pre-packed in a column?
No. The resin comes as a 4mL, 50% slurry that you pour and pack/equilibrate yourself. As a convenience we include a disposable column with a frit, a stop and a lid.AbCA
Does the AbC recognize AviTag™ if it is fused in tandem with a His-Tag?
Yes, and the order does not matter. It can be AviTag™-His-Tag or His-Tag-AviTag™.AbCA
Does the AbC recognize both N-terminal and C-terminal AviTag™ protein fusions?
The AbC recognizes only the C-terminal AviTag™ fusion presentation.AbCA
Is the AbC monoclonal or polyclonal? What species was it produces in? What form of IgG is it?
The AbC is mouse monoclonal IgG2B.AbCA
Does the AbC recognize the biotinylated AviTag™ as well as the unbiotinylated form?
Gaussia Luciferase protein
How much Coelenterazine (CTZ) do I have to use?
SA-GLuc is using a mutated version of Gaussia that has a higher and prolonged turnover rate. In order to use its full potential we recommend a final CTZ concentration of 100 µM. Coelenterazine can be purchased here: https://www.avidity.com/avitag/ctz-1Gaussia Luciferase protein
How should I dilute SA-GLuc?
You can use TBS, pH 7.4-7.8 for the dilution of SA-GLuc. However, we are recommending Avidity’s Gaussia Dilution Buffer for increased protein stability and activity.Gaussia Luciferase protein
Why I’m not able to transform these cells with my plasmid of choice?
AVB100 are not made competent. Please use established protocols to make them competent or purchase electro-competent AVB100 cells, called EVB100.AVB 100
Will AVB100 grow on LB-plates with antibiotics?
The untransformed cells will not grow if any antibiotics are present. These cells do not carry any antibiotic resistance, unless transformed with a plasmid (e.g. pAN vectors with Ampicillin resistance).AVB 100
Why do I get a low yield from my plasmid prep?
This is a low copy number plasmid. Please use rich media like TYH or TB and a higher volume (5-10 ml for a mini prep) to increase the plasmid yield.AVB 99
What antibiotic do I have to use?
Please use Chloramphenicol at a concentration of 10 μg/ml to maintain the plasmid.AVB 99
Why is chloramphenicol not added to the growth/induction media to maintain the BIP-300 plasmid when it is used in the initial overnight culture? (relevant only if AVB101 E. coli is used as the host strain)
We find that the chloramphenicol is not necessary at this point to maintain the pBirAcm BirA over-expression plasmid. It is well enough maintained because of its low copy number.BIP-300
Do your vectors work off the T7 promoter expression system?
No. The BIP-300 positive control plasmid (and the pAN /pAC vectors) works off the Trc promoter.BIP-300
How do you remove the excess free d-biotin?
We have found that using a simple desalting column is effective at removing the d-biotin since it is so small (244.3 Daltons).BirA
Is it necessary to quench the reaction or remove the BirA when the reaction is done?
No. The reaction is basically self-quenching. As the biotinylation nears completion it slows down as the BirA “searches” for unbiotinylated substrate. As long as there is ATP and d-biotin available and the ATP has not been exhausted, the reaction goes to completion. The BirA afterwards is inert. It will not react with anything else besides ATP and substrate. You can remove the BirA with size-exclusion chromatography or a cationic exchange resin (assuming your protein does not also stick to it) if you desire but it is not necessary.BirA
How long can I store the BirA enzyme and still maintain full functionality?
Our BirA enzyme is a very stable protein. It can be stored for years at -80 degrees Celsius and still have full functionality (only if continuously frozen!). Once thawed, unused portions can be flash-frozen in liquid nitrogen and stored again at -80 degrees Celsius with only a minimal loss of functionality (10-20% each time). If stored at 4 degrees Celsius after thawing, the BirA enzyme is functional for weeks. Count on perhaps a 5-10% loss in activity per week at 4 degrees Celsius. That being said, the most volatile part of the BirA biotinylation reaction kit is the BiomixB. BiomixB contains ATP which is subject to hydrolysis when not frozen. Generally when we are contacted about problems with the BirA reaction it is because the BiomixB has gone off. All is not lost however as additional ATP added to the reaction will start it back up again.BirA
Do I need to add additional d-biotin to the reaction mix?
Usually not. The BiomixB included in the BirA biotinylation reaction kit contains 500uM d-biotin for 50uM final concentration. The BIO200 (500uM d-biotin) also included in the BirA biotinylation reaction kit is for use with modified amounts of substrate in the biotinylation reaction. If you want to try to biotinylate larger amounts of substrate or greater than recommended concentrations, extra d-biotin (and extra BirA) can help decrease reaction times.BirA
How much BirA do I need to biotinylate my AviTag’d protein?
The long answer to this question is that for every 10 nmol of substrate (at 40uM concentration in the final reaction mix) we recommend 2.5ug of BirA enzyme to complete the biotinylation in 30- 40 min. at 30°C. The short answer is we recommend about 1ug of BirA to every 100ug of AviTag’d peptide/protein for good rates of biotinylation. Enzyme dynamics are complicated in that lower biotinylation substrate concentrations (or lower BirA concentrations) increase reaction times significantly. For example, where 40uM of substrate may take 30 minutes to biotinylate to completion, 4uM substrate concentration may take 5 hours with the same amount of BirA. The best reaction conditions will be determined by the individual user.BirA
What is the molecular weight of the BirA enzyme?
Does the BirA contain an affinity purification tags, such as the His-tag?
No. Our BirA is the wild-type purified via traditional protein purification methods from E. coli. BirA purified in this manner without fusion tags is the most active available.BirA
What are the best storage conditions for birA?
The birA enzyme arrives frozen. If you are not going to use it immediately, then store it at -80°C. Once the enzyme has been thawed, it should then be stored at 4°C. We do not recommend repeatedly re-freezing the enzyme as there is a 5-10% loss of activity during each freeze/thaw cycle. At 4°C the enzyme is stable for several months. For long-term storage, the enzyme can be safely re-frozen by dropping into liquid nitrogen before storing at -80°C.BirA
Do detergents inhibit birA?
We have tested Tween 20 at 0.1% concentration and not found any effect on the biotinylation reaction.BirA
Does your birA enzyme contain proteases?
The level of protease contamination of the birA enzyme is almost undetectable. If the target protein has not been extensively purified, then this is a much more likely source of proteases! If you are finding proteases to be a problem, we recommend adding a protease inhibitor cocktail. For proteins expressed in E. coli we recommend the Sigma protease inhibitor cocktail (catalog #P8849) at a 1/100 dilution. For mammalian or insect cell expression, we recommend a protease inhibitor cocktail containing leupeptin, pepstatin and PMSF to the biotinylation reaction mix.BirA
Why is my expressed protein not biotinylated?
Make sure you added 0.2-0.4% L-arabinose to the media to induce overexpression of the BirA protein, necessary for in vivo biotinylation. We also recommend supplementing the media with additional biotin with a final concentration of 50 μM. Biotin solution is available here (link to biotin product).EVB 100
What antibiotic do I have to use?
The EVB100 strain itself is not carrying any antibiotic resistance. The plasmid that you are using for electroporation will carry the antibiotic resistance. For example Ampicillin for AviTag pAN and pAC vectors.EVB 100
For standard transformation we heat shock at 42C for 30 sec. The tubes we usually use are not the same type as you supplied the cells in. Should I transfer the cells to a more usual tube we use for these procedures or use your tube?
For higher transformation efficiency we recommend using thin walled tubes to get an optimal heat transfer. We guarantee a transformation efficiency of 106 cfu/μg in the tubes that we provide. If you are using a water bath for heat shock please make sure that no air is trapped underneath the tube. Tilt the tube to remove any air.CVB 101
What is the amino acid sequence for your MBP-AviTag fusion protein?
The sequence is provided below. MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLA EITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTW PLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNI DTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPR IAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGSLSTPPTPSPSTPPTGLNDIFEA QKIEWHE Color legend: MBP-IgA hinge linker-AviTagBIS-300
Is the BIO-500 sterile?
Yes. The BIO-500 d-biotin solution is filtered for sterility through a 0.22μm filter before it is packaged.BIO-500
Do I need to purchase the BIO-200 500μM d-biotin solution separately from the BirA 500/Bulk BirA reaction kits?
Usually not. Both kits come with vials of BIO-200 already included in them. Plus, the BiomixB included in the kits contains enough d-biotin for the typical BirA biotin-protein ligase reaction. Additional d-biotin may be added if desired but is usually not necessary.BIO-200
What vectors are compatible with AVB101?
Most expression vectors that are used in laboratories today are derive from the colE1 plasmid which includes pBR322, pUC, pGEM, pBAD and are compatible with AVB101. If you want to avoid a two plasmid system you could also use AVB100 where the BirA encoded on the bacterial chromosome and is expressed via the arabinose promoter.AVB 101
Biotinylation of Proteins
Can I achieve biotinylation of proteins bound to a nitrocellulose membrane?
This is something we have not tried. Please let us know if you have any experience with this kind of experimental set-up.Biotinylation of Proteins
I am preparing my own biotin solutions. Why am I having problems with the biotinylation reaction?
We have noticed that the d-biotin from some suppliers tends to 'go off' rapidly. We routinely used d-biotin from Research Organics, as this was the most stable we have come across. Unfortunately, this d-biotin is no longer on offer so we have switched to Sigma-Aldrich d-biotin, specifically catalog #B4501-10G. This d-biotin seems to be just as stable in solution and we are having good luck with it to date.Biotinylation of Proteins
How can I quantitate the level of biotinylation of my protein?
We have developed a simple 'dot blot' assay and plate assayBiotinylation of Proteins
What percent of my protein can I expect to be biotinylated in vivo?
Using an E. coli strain that over-expresses birA such as AVB101, we see 85-95% biotinylation of substrate proteins.Biotinylation of Proteins
Can the in vitro biotinylation be scaled up?
Yes. Milligram quantities of protein can be biotinylated in overnight reactions. A protocol is given in Crawford et al., Immunity 8, 675-682, 1998.Biotinylation of Proteins
I am using the consensus peptide described by Schatz (1993) but the levels of biotinylation are poor. How can I improve this?
The consensus sequence is, in effect, an average of the efficient and inefficient peptide sequences. The most efficient sequence is #85. (G L N D I F E A Q K I E W H E) You need to convert over to this.Other