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).

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.

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.

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.

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.

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.

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.

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.

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.

What is the molecular weight of the BirA enzyme?

33.5 kDa.

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.

What is the molecular weight of the BirA enzyme?

33.5 kDa.

What is the amino acid sequence for your MBP-AviTag fusion protein?

The sequence is provided below.

MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLA
EITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTW
PLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNI
DTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPR
IAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGSLSTPPTPSPSTPPTGLNDIFEAQKIEWHE

Color legend: MBP-IgA hinge linker-AviTag

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.

What is the molecular weight of the BirA enzyme?

33.5 kDa.

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.

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.

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.

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.

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).

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.

What is the molecular weight of the BirA enzyme?

33.5 kDa.

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.

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.

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.

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.

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).

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.

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.

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.

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: