Biotinylation of Targets

For those who need to biotinylate your proteins, here's the protocol. Gwen will have it on Blackboard as well.

Biotinylation of Protein

The N-hydroxysulfosuccinimide (NHS) ester group on this reagent reacts with the e -amine of lysine residues to produce a stable product. Although a -amine groups present on the N-termini of peptides react with NHS esters, a -amines on proteins are seldom accessible for conjugation.

NHS esters react with deprotonated primary amines, therefore, the reaction requires neutral to basic pH values to proceed. Primary amines react with NHS esters by nucleophilic attack and NHS is released as a byproduct. Hydrolysis of the NHS-ester competes with the reaction in aqueous solution and increases with increasing pH.

With large proteins, labeling of several lysine residues and the N-terminus does not usually harm protein function or binding properties. With short peptides, however, the random biotinylation of the ε-amino groups of lysine residues is much more likely to block binding sites whose function is necessary for downstream applications of the biotinylated peptide. Labeling of lysine residues in peptides, especially short ones, may alter their functional properties and/or immunoreactivity.

Preparing the Target

1. Find out the molecular weight of your target. Most range between 2000 and 70,000 Daltons. A Dalton is a measure of grams/mol or ug/umol.
1. If your target is more than 1mg in the vial, then also find out the extinction coefficient.
2. If it is under 1mg (say 100ug?), then determine if it is already resuspended in a buffer.

i. If it is in solution determine what the solution is. Tris buffers have primary amines and react with the biotinylation reagent.

2. Calculate the molar concentration or quantity of your target.
1. i.e. if you have 100ug of target and it is 34kDa, then (100ug / 34000ug/umol) = 0.00294umol or 2.94nmol or 2940pmol total.
2. If the above protein was in 100ul it would be at a concentration of 2940pmol/100ul = 29pmol/ul or 29uM.
3. If your protein is in powder form (desicated), then resuspend it in a suitable volume of buffer.
1. Typically, you should consider between 20 and 200uM concentration. You can certainly go higher, but try not to go much lower.
2. 100 to 200ul of a 1X PBS buffer or 50mM HEPES buffer work well. Try to use the buffer (not with salts added) that you plan to perform the selection with.
3. DO NOT use Tris buffer.
4. Calculate the volume of target to biotinylate.
1. Typically you should biotinylate between 1000 and 4000pmol protein total. However, please don’t biotinylate all of your protein in case something goes horribly wrong. In the above example, I would suggest biotinylating 1500pmol. 1500pmol / 29pmol/ul = ~51ul or half the total volume.
5. Aliquot any remaining protein in easy use volumes in 0.2ml tubes to prevent freeze thawing multiple times. Label the tube with the protein name.
1. Typically aliquots will be the same volume as used for step 4.
6. Label a sticky note with the target name, supplier, stock number, lot number, concentration and volume per tube. Also put your name and the date it was first aliquoted.
7. Place aliquots in the -80°C freezer.
1. Mark the empty supplier tube with an X on the cap and place it in the freezer.
2. In addition, put the sticky note in the box.

Preparing the Biotinylation Reagent

8. Remove EZ-Link Sulfo-NHS-LC-Biotin (Pierce product 21335) from the desiccator (blue saucer) in the -20 °C freezer box and allow it to warm to room temperature (5 minutes).
9. Add 2 mg of EZ-Link Sulfo-NHS-LC-Biotin to ~200 uL of 50mM HEPES, pH 7.5.
1. MW of Biotin is 556 g/mol. 2 mg gives 3.6 umole reagent. If mixed in 200ul buffer, the concentration is 18nmol/ul.
10. You should add between 5x and 10x EZ-Link reagent to target. Because the reagent starts to hydrolyze once its dissolved, it is active for only a short period of time.

Biotinylating the Target.

• The reagent will attack primary amines on the protein (or buffer if using Tris). These are found at the N-terminus (NH₂) or lysine residues.
• If you use too much reagent, the protein will denature when immobilized to the bead.
• If you use too little reagent, some protein molecules may not be biotinylated and therefore may be lost during washes.
• It may be necessary to choose multiple molar excesses and test each for protein function/binding after biotinylation with some known property.

11. Add X ul of step 4 to Y ul EZ-Link reagent from step 6. This is your total Z ul volume.
1. In our example, we should add 51ul protein (volume X) to 1ul EZ-link reagent (volume Y) for 9x molar excess.
2. The unused Biotin solution should be discarded. Try to biotinylate with others as this reagent is expensive and much is wasted.
12. Refrigerate the reaction at 4 C for >2 hours – overnight.
1. Shaking or agitation during incubation is prefered but not required.

Purifying the target.

• It is important to remove any unbound biotin from the reaction. Therefore a desalt column should be used with a molecular weight cutoff below your target.

Zebra Colum Sizes and recommended sample volumes
Column Size	Resin Bed	Sample Size
Micro	75ul	2-12ul
0.5ml	0.5ml	30-130ul
2ml	2ml	200-700ul
5ml	5ml	500-2000ul
10ml	10ml	1500-4000ul

• The NHS-Biotin reagent has a molecular weight of » 600 Da. The Zebra spin columns have a MWCO of » 7000 Da (anything smaller will be trapped).
• Sephadex can also be used. G-10 has a MWCO of ~ 700Da. G-25 has a MWCO of ~ 5000Da, G-50 has a MWCO of 30kDa and G-100 has a MWCO of 100kDa.
• Use the proper column volume for your total reaction (see table).

13. Measure the absorbance of reaction on the nanodrop using the “Protein A280” button. Print out the graph.
14. Follow the desalting protocol on Blackboard with half your sample.
15. Remeasure the absorbance and compare the A280 measurements before and after.
16. If the second measurement is within 50% of the first, desalt the remaining protein over the same column.
17. Discard the desalting column after use.

Prepping the Target for Immobilization.

• Provided sufficient labeling of protein, you should have recovered it all in the volume of buffer your initial reaction (Z ul, step 8).

18. Calculate the concentration of your protein. You can do this spectrophometrically if you know the extinction coefficient, or assume a rough concentration.

1. Your concentration of protein should be Q pmol used for for biotinylation (from step 4) / Z ul biotinylation reaction.
2. From the example above, we had 1500pmol in a volume of 51ul. 1500pmol / 51ul » 30 pmol/ul or uM.

19. If you lost some sample in the desalt procedure (your final A280 measurement is significantly less than your initial A280 reading) then you must adjust by first calculating the difference and multipling through by that factor.
1. For instance, if the initial measurement was 22.5 and the last measurement is 18.7, then you recovered 18.7/22.5 or 83%. Assuming you initially biotinylated 1500 pmol then you recovered .83*1500 or roughly 1250 pmol.
20. Aliquot your protein into tubes for long term storage. Label the tube with the protein name-Biotin.

3. Aliquot 100pmol target per tube.
4. In our example we would use ~ 4ul per tube or 12 tubes.

21. Label a sticky note with the target name-Biotin, date the reaction was performed, concentration volume and your name.
22. Place aliquots in the -80°C freezer in the same box as the unbiotinylated target.

5. In addition, put the sticky note in the box.
6. Mark where your protein is on the freezer map on the door of the -80 °C.