UsingImmobilized Streptavidin

David Wilson


Strategic Planning and Product Information:

Streptavidin binds to biotin with extremely high affinity (Kd=10-15M),and the protein is very stable under a number of normally denaturing conditions.This makes this system very powerful, since a protein or RNA that containsbiotin can be immobilized on the column and washed under fairly extremeconditions, thus removing >99.7% of the unbiotinylated molecules. The affinityis very strong and stable over long periods of time. For more informationconsult the product sheet for the Pierce Immunopure Immobilized Streptavidin,product number 20349. Pierce also sells streptavidin immobilized on anothermatrix. This product is called Ultralink Immobilized Streptavidin. Thismatrix has higher capacity. I do not use it, however, because, when I heatthis matrix to 95 degrees for 5 minutes to release immobilized RNA, theRNA is degraded. Also availible from Dynal and other companies is straptavidinlinked to magnetic beads. This matrix is easier to use, gives less background,but is much more expensive and has lower binding capacity, so it can onlybe used for relatively small-scale. Also, heating these magnetic beadscauses release of iron, which degrades the RNA. Thus, the protocols listedbelow are for the Pierce Immunopure Immobilized Streptavidin, product number20349.

The capacity of the matrix varies slightly from lot to lot, but is generallyabout 75nmoles biotin bound per ml of beads (note that this volume referesto the volume taken up by the beads plus solvent after the beads have settled).If you wish to use the material at or near its capacity, it is best tomeasure the capacity yourself.

The matrix has a fairly high non-specific RNA binding activity, butthis can be greatly reduced by pre-incubating the non-specific RNA (egtRNA) and washing under denaturing conditions.

Because the streptavidin-biotin interaction is so strong and stable,it is very difficult to remove biotinylated molecules from the matrix.However, this can be accomplished by heating in the presence of biotin(see below).

Reagents required:

3M NaCl
5% NP-40 (detergent)
0.5M EDTA pH 8
tRNA or other non-specific competitor RNA (~1mg/mL)

Solution A:

1M NaCl
20mM Tris pH 7.3
5mM EDTA pH 8
0.1% NP-40

Solution B: 

2mM Tris pH 7.3
0.5mM EDTA pH 8
0.1% NP-40

Solution C:

4M Urea
10mM Tris pH 7.3
1mM EDTA pH 8
0.1% NP-40

Solution D:

2mM Tris pH 7.3
0.5mM EDTA pH 8
Biotin solution:
use Sigma biotin, MW = 244g/mol
solution is 2.5mM biotin

biotin is an acid that has low solubility in water. After adding biotinto water, titrate in Tris base until the pH reaches 7.4. At this pointall the biotin should go into solution. Also add 1mM EDTA.

Sample protocol: Selecting biotinylated from non-biotinylated RNApool molecules.

Preparation of Sample: This protocol was used to select thosepool RNA molecules that covalently linked themselves to a biotinylatedsubstrate RNA, thus themselves becoming biotinylated. Fifty pmol of poolRNA (MW=75,000Da) and 100pmol of biotinylated substrate were incubatedin a buffer that contained 200mM KCl and 60mM MgCl2 (the enzymatic conditions).Then, to prepare the sample for loading onto the column, EDTA was addedto chelate the Mg++, the NaCl concentration was raised to 1M, and the samplewas broght to 0.1% NP-40 (a non-ionic detergent). To remove any aggregatedRNA from the sample before loading it onto the column, it can be spin-filtered(e.g. Millipore cat No. UFC30GV00) immediately before adding to the matrix.

Preparation of matrix: The commercial bottle of streptavidin-agarosewas agitated to resuspend the matrix. Seventy ul of the slurry was removedand placed into a disposable Bio-rad drip column. This was washed with10mL solution A (1ml was added, allowed to drip through, then a secondmL was added, etc.). The beads, still in the column, were respuspendedin 400ul of buffer A, and the slurry was transferred to a 1.5mL Eppendorftube. Two ug of tRNA was added, and the mixture was nutated for 30 mintesat room temperature.

Biotin Selection:

Add the sample to the matrix, and allow to nutate 30 more minutes. Addthe slurry to a new BioRad column. After the bed has settled (~5minutes),open the stop-cock and allow column to drip.

Wash with 1ml (~30 column volumes) of buffer A

repeat 2 more times

Wash with 700ul (~30 column volumes) of buffer B

repeat 5 more times

Wash with 700ul (~30 column volumes) of buffer C

repeat 5 more times

Wash with 700ul (~30 column volumes) of buffer D

repeat 5 more times

Cap column, add 450ul buffer D, resuspend and remove (with a P1000)as much slurry as possible,and transfer it into PCR tube

Spin PCR tube for ~1minute and discard supernatant

To any remaining beads, add another 450ul buffer D to column, remove,resuspend beads and transfer slurry to same PCR tube. Discard column.

Spin PCR tube for ~1minute and discard supernatant.

Add 80ul of the biotin solution

Heat at 95C, 4 minutes (in PCR machine, for example).

Transfer slurry to spin filter (e.g. Millipore Ultrafree-MC 0.22um filterunit, cat No. UFC30GV00). Spin 1 minute. Keep flow-through. Do phenol/chloroformextraction

Wash the beads with 100ul solution D

Do chloroform extraction

repeat 2 more chloroform extractions

Add salt and glycogen, and EtOH to precipitate RNA. 

Pitfalls and troubleshooting:

This selection method results in 40-80% recovery of biotinylated RNAand less than 0.3% background sticking by unbiotinylated RNA. Omiting eitherthe pre-incubation of the column with tRNA or the wash with the urea-containingsolution and low salt solutions would increase the background. In generalthe organic extractions after column elution are not necessary, but theymay help with reproducibility. There do appear to be some inhibitors ofthe RT reaction that are eluted from the column using this procedure, suchas tRNA, biotin, and possibly inhibitors released from the agarose beads.However, if you follow this protocol as specified, with the amount of tRNAspecified, and RT the entire sample in a 50ul reaction using the standardreaction procedure (see other section), high yields can be obtained. Whenusing larger volumes of beads, and/or larger amounts of tRNA or biotin,it may be necessary to remove inhibitory contaminants from the sample ordilute it into a larger RT reaction.

Attachment: Streptavidin-agarose product sheet from Pierce.