1. MACS® Technology for magnetic isolation of cells and molecules
Thuesday:
MACS® Technology for magnetic isolation of cells and molecules
Introduction
Features of paramagnetic MicroBeads
General procedure of magnetic cell isolation
Overview of applications in molecular biology
µMACS epitope tagged protein isolation
Expression of tagged/fusion proteins, e.g. GFP fusion proteins
Magnetic protein isolation
Wednesday:
Detection of proteins, results and trouble-shooting
Optimizing protein expression analysis by transfected cell selection (MACSelect) 1

2. Expression of tagged/fusion proteins, e.g. GFP fusion proteins
organism for protein expression (bacteria, eukaryotic)
expression vector (promoter etc.)
transient or stable transfection
choice of tag 1

3. General features of the MicroBeads
Super-paramagnetic
Colloidal non-sedimenting Extremly fast reaction kinetics & efficient
Extremely small magnetic labeling
Biodegradable & non-toxic
The MicroBeads are super-paramagnetic, meaning they are not magnetically by itself. Additionally, they form a stable colloidal suspension and don‘t sediment or aggregate in magnetic fields. Due to their extremely small size (approx. 50 nm in diameter) they provide a high surface-to-volume ratio. They are biodegradable and non-toxic. Altogether this leads to extremly fast reaction kinetics and a very efficient magnetic labeling with high yields.

4. Magnetic protein isolation
Cell lysis
Magnetic labeling with MicroBeads
• µMACS Protein A and Protein G MicroBeads
• µMACS Anti Epitope Tag MicroBeads, e.g. Anti-GFP MicroBeads
• µMACS Streptavidin MicroBeads
3. Specific protein isolation with µ or M Columns and MACS® Separator 1

5. Cell lysis and immunolabeling SDS-PAGE or enzymatic assay
Immunoprecipitation with µMACSTM Protein A / Protein G MicroBeads
Pure protein in less than 2 hours
Starting from isolated or cultured cells, a cell lysis is performed with subsequent removal of cell debris.
Antibody and Protein A/G MicroBeads are added and incubated for approx. 30 min, time only depending on the antibodies specificity. No pre-absorption and overnight incubation is necessary.
The lysate is applied on the column placed in a magnetic separator. While the protein of interest bound to the specific antibody and the MicroBeads are retained in the magnetic field of the column, unspecific proteins and other molecules are easily washed away. With the MACS Column Technology, washig steps are very efficient and convenient.
Finally, the protein is eluted together with the antibody with hot SDS-Gel loading buffer for SDS-PAGE or an enzymatic assay is performed on the column. |[For the enzymatic assay enzymes and appropriate buffers are applied on the column where the protein is still bound. Incubation is then performed in a reaction volume of 50 µl at the necessary temperature. For incubations of 37 or 42°C the thermoMACS - a heatable µMACS separator (or an incubator) can be used. Then after appropriate washing only the activated substrate is eluted, which can be measured i.e. by PAGE gel.]
The complete immunoprecipitation takes hrs instead of days.
Cell lysis and immunolabeling
Magnetic separation
SDS-PAGE or enzymatic assay

6. Immunoprecipitation with µMACS
IP of SV40 large T antigen from COS cells with µMACSTM Protein G MicroBeads
Silver stained SDS PAGE
L: Lysate
M: Marker
Lane 1: Large T antigen (indicated by arrow)
Lane 2: Isotype control
Lane 3: Negative control
Example of an IP of the large T antigen. COS cells (overexpressing large T antigen) have been lysed and IP performed with anti-SV40 large T antigen antibody and Protein G MicroBeads.
Silver stained SDS PAGE
Lane 1: large T antigen (≈90 kD)
Lane 2: isotype matched control antibody (rat anti-mouse antibody)
Lane 3: negative control without antibody
Lane 1+2: antibody heavy chain (≈50kDa), light chain (≈25 kDa)

7. Immunoprecipitation of surface antigens
IP of biotinylated surface antigens of B cell line JOK-1 with µMACSTM Protein A
Western Blot with Streptavidin-HRP
Lane 2: anti-CD22 (HD 239)
Lane 4: anti-MHC class I (W6/32)
Lane 1 and 3: isotype matched antibody as control.
CD 22 (140 kDa)
MHC Class I (45 kDa)
Customer report (flyer available)
Intakt JOK-1 (lymphoblastoid B cell line) cells were biotinylated, so that all surface markers are biotinylated. Cells were lysed and the lysate incubated with a specific antibody against either CD22 or MHC class I (surface molecules) and Protein A MicroBeads. After IP the eluate was separated in a SDS-PAGE. The following Western Blot was performed with Streptavidin-Horse Radish Peroxidase to detect the biotinylated surface molecules. No background is visible due to efficient washing on the column.

8. Co-IP of the androgen receptor and it´s target
Western Blot of AR-co-immunoprecipitated Beta-Catenin using anti-Beta-Catenin antibody
Western Blot of Immunoprecipitated AR using Anti-AR antibody
Customer report (flyer available)
Intakt JOK-1 (lymphoblastoid B cell line) cells were biotinylated, so that all surface markers are biotinylated. Cells were lysed and the lysate incubated with a specific antibody against either CD22 or MHC class I (surface molecules) and Protein A MicroBeads. After IP the eluate was separated in a SDS-PAGE. The following Western Blot was performed with Streptavidin-Horse Radish Peroxidase to detect the biotinylated surface molecules. No background is visible due to efficient washing on the column.
AR was immunoprecipitaed from DHT-stimulated (lane 1, 3) or unstimulated LNCaP cells (lane 2, 4) with µMACS Protein G MicroBeads (lane 1-2) or with Protein A/G agarose beads (lane 3-4).
Courtesy of D. Mulholland, Vancouver, Canada

9. ChIP - Chromatin immunoprecipitation
PCR of the PSA gene using ChIP obtained DNA
IP was carried with µMACS Protein G MicroBeads (lane 1-2) or with Protein A/G agarose beads (lane 3-4). Anti-AR antibody (lane 1, 3) or as a control anti-IgG (lane 2, 4) were used for Chip.
Courtesy of D. Mulholland, Vancouver, Canada

10. New µMACSTM Epitope Tag Protein Isolation Kits Application
isolation of recombinant proteins with epitope tags and their interaction partners from eukaryotic cells
The new µMACS Epitope Tagged Protein Isolation Kits allow a very pure isolation of recombinant proteins with epitope tags from eukaryotic cells. Additionally, the isolation of interacting proteins or complete protein complexes is possible.
The MicroBeads are coupled to highly specific monoclonal antibodies against HA (hemagglutinin), 6xHis, c-myc or GFP (Green Fluorescent Protein) tags.
The kits contain lysis, wash and elution buffers optimized for mammalian cells, but can also be used for yeast or bacteria cells.

11. µMACSTM Epitope Tag Protein Isolation Kits
Pure proteins in less than 2 hours
• highly specific MicroBeads coupled to monoclonal antibodies • eliminate background with effective column washing
• optimized buffer set, small elution volume
• MACS® Columns for easy handling
As the antibody-tag binding is very specific and sensitive, even minimal amounts of protein can be isolated out of a large background of unspecific proteins. This allows the isolation of recombinant proteins from eukaryotic cells, which is e.g. for His tagged proteins hardly possible with any IMAC resin due to the large amount of histidine rich proteins in eukaryotic cells.
The small elution volume of 50 µL allows direct loading of the protein on a PAGE gel.
The complete procedure is -due to MACSTechnology- very fast and handling is very easy.

12. µMACS Epitope Tagged Protein Isolation Kits
Epitope-tags
His : artificial tag of 6 Histidines
HA= Hemaglutinin (or Hemagglutinin) : viral gene
c-myc : human c-myc is proto-oncogene
GST = Glutathion S-transferase
GFP = Green Fluorescent Protein
BM 10/2002 1

13. MACS in Molecular Biology
GFP = Green Fluorescent Protein from Aequoria victoria
BM 10/2002 1

14. MACS in Molecular Biology
Epitope tagged proteins - Advantages:
study of proteins possible without a specific antibody (production of specific Ab difficult & time-consuming)
simplified isolation: known epitope + known interaction
standardized isolation: using one established system (expression vector/tag) for study of different proteins
BM 10/2002 1

15. µMACS™ Epitope Tagged Protein Isolation Kits
Standardized isolation of different proteins
recombinant proteins A-C isolated with protein specific Ab
tagged recombinant proteins A-C isolated with tag specific Ab
BM 10/2002 1

16. MACS in Molecular Biology
Epitope tagged proteins: working areas - examples
DNA sequences (e.g. identified by HUGO =human genome organization) give no information about
structure
binding partners
function
cellular localisation
they need to be expressed in eukaryotic cells
BM 10/2002 1

17. µMACSTM Epitope Tag Protein Isolation Kits
Working scheme for isolation of tagged proteins
First a cell lysate is prepared, cell debris removed by centrifugation, and MicroBeads are added. Magnetically labeled cell lysate is tehn applied on the µ Column. Unspecific molecules are then washed off while the MicroBead-Epitope-Tag-Protein remains in the magnetic field of the column. The target protein can finally be elutedwith hot SDS-gel-loading buffer: In a similar procedure interacting proteins or whole protein complexes can be purified.
Lyse cells and add MicroBeads
Apply to MACS Column
Remove unbound material by stringent washing
Elute target protein while MicroBeads remain on column 1

18. Protein isolation with µMACSTM Anti-c-myc MicroBeads
Isolation of c-myc-BDCA2 from biotinylated cell lysate
Western Blot anti-c-myc/POD
Lane 1 -3: 10, 20, 50 ng of Multi-Tag Protein
Lane 4: c-myc-BDCA2 (1/25 eluat)
Lane 5:c-myc-Dectin2 (1/125 eluat)
Lane 6: c-myc-BDCA2 (1/50 eluat)
Lane 7: c-myc- Dectin2 (1/250 eluat)
Cell source:
5x 10E6 293 T HEK (human embryonal kidney)
40 kDa -
30 kDa -
24 kDa -

19. 35S-methionine labeled isolation
Isolation c-myc-BDCA-2 using anti-HA and anti-c-myc MicroBeads
35S-methionine labeled isolation
anti-HA anti-c-myc
c-myc-BDCA-2
This is the result of the BDCA-2 expression:
The picture on the left side shows a western blot with an anti-HA antibody (+ anti-mouse-HRP) and peroxidase catalysed staining. Without transfected cell and protein isolation hardly any BDCA-2 can be detected on the western blot. With µMACS HA-Tag protein isolation a strong protein band is visible. The extended band is due to glycosylation of BDCA-2 resulting in different sizes. Additional bands show a dimer, signals below belong to BDCA-2 degradation products.
The combination of MACSelect and µMACS protein isolation gives even stronger bands. Now you don‘t have to scale up transfection and immunoprcipitation in case of low transfection efficiencies and low protein expression.
(10^7 cells each, no isolation: lysis in 50µl, 1/5 on gel; Tag-isolation: 1/5 eluate; MACSelect+Tag isolation: 10^8 cells transfected, approx. 50% => Tag isolation from 5x10^7 cells
(HA-tag control protein: 8.5ng, 17.5ng, 51ng; ECL develop ~1min; no antibody light chain visible- only with very long exposure)
The x-ray picture on the right hand side shows the specific isolation of c-myc-BDCA-2, that is not detectable with HA-Tag isolation.
© Miltenyi Biotec

20. µMACSTM Streptavidin Kit
Application
isolation of any biotinylated molecules like DNA, RNA, proteins, etc. and their interacting molecules
• Isolation of DNA-binding proteins (transcription studies)
• Isolation of RNA-binding proteins (translation studies)
• Isolation of (specific) transcripts (e.g. viral transcripts)
• Phage display
• Subtractive hybridisation
• SAGE
• Isolation of tRNA molecules
The µMACS Streptavidin Kit allows the isolation of biotinylated molecules and their interaction partners. Isolated molecules can be nucleic acids like DNA or RNA or proteins, peptides, protein complexes, etc.
starting protocols
available on webpage

21. µMACSTM Strepavidin Kit
Isolation of DNA-binding proteins
Isolation of DNA binding proteins as an example of an application of the µMACS Streptavidin MicroBeads:
Cells are lysed and the lysate is incubated with a specific biotinylated DNA probe. Incubation time is dependent on affinity of the specific molecules. Then µMACS Streptavidin MicroBeads are added, there is virtually no incubation time necessary. Now a complex is formed between the specific DNA-binding protein and the biotinylated DNA bound to the Streptavidin MicroBeads.
The whole probe is applied on the column and unspecific bound molecules removed by stringent washing (for proteins higher concentration of salt leads to more specific binding; for nucleic acids the specifity is enhancd by decreasing salt concentration).
Finally, the column technology allows release of the target molecule only, by choosing an appropriate buffer. Here only the pure DNA binding protein is eluted while the DNA probe (due to the strong interaction of biotin and streptavidin) remains on the column.
Isolation of target molecules with biotinylated probes, here the isolation of DNA-binding protein. Incubate your sample of interest with biotinylated DNA probe (1), add µMACS Streptavidin MicroBeads (2) and apply to column (4). Wash to remove unbound material (5) and elute your target molecule while the biotinylated probe remains on the column (6).

22. Transcription control studies with µMACSTM Streptavidin Kit
Purification of DNA binding proteins
IL non
Marker stimulated stimulated
Western blot of Stat6 transcription factor. Dermal fibroblasts were stimulated with IL-4 and their nucleus extracts were incubated with biotinylated DNA already coupled to µMACS Streptavidin MicroBeads. The isolated binding proteins were purified and proofed by SDS-PAGE and Western blotting
 Stat6
If IL-4 binds to the surface marker of cells, it induces a signal cascade, which finally leads to the activation of a transcription factor. This transcription factor will then enter the nucleus and induce the expression of certain genes.
In this experiment the nuclear extract of Il-4 stimulated fibroblasts was incubated with the specific DNA-probe for the Stat6 transcription factor. The probe was bound to µMACS Streptavidin MicroBeads. The isolated DNA binding proteins where shown in a Western blot with a Stat-6 antibody. Only in stimulated cells the transcription factor is found in the nucleus.

23. Isolation of a DNA binding protein
Using biotinylated target DNA for LNCaP nuclear protein associated with PSA promotor region
2D gel electrophoresis of protein bound to the biotinylated target DNA (A) or a non-specific biotinylated control DNA (B)
Courtesy of D. Mulholland, Vancouver, Canada
Customer report (flyer available)
Intakt JOK-1 (lymphoblastoid B cell line) cells were biotinylated, so that all surface markers are biotinylated. Cells were lysed and the lysate incubated with a specific antibody against either CD22 or MHC class I (surface molecules) and Protein A MicroBeads. After IP the eluate was separated in a SDS-PAGE. The following Western Blot was performed with Streptavidin-Horse Radish Peroxidase to detect the biotinylated surface molecules. No background is visible due to efficient washing on the column.

24. Isolation of a transcription factor
from E. coli expressing the recombinant factor M
Coomassie Blue-stained PAGE gel
E.coli extract was incubated with a biotinylated operator followed by trapping the protein-DNA complexes to streptavidin
Lane 1: flow-through
Lane 2: eluate
30 kD
transcription factor
A regulatory protein, expressed in Escherichia coli, was purified in a single step from a concentrated crude extract of induced E. coli (E. coli strain JM109).
Purification was accomplished by incubating the extract briefly with a biotinylated operator DNA followed by the addition of mMACS Streptavidin MicroBeads to bind the protein-DNA complexes. The mixture was applied to a column. After removal of unbound and non-specifically bound material by stringently washing the column, the regulatory protein was eluted in high-salt buffer (second through fifth drop to emerge from the column) and shown to migrate as a single Coomassie Blue-stained band of the expected monomer molecular mass (30-kDa) after denaturing polyacrylamide gel electrophoresis.
The molecular identity of regulatory protein was further confirmed by automated Edman degradation of the polypeptide’s N-terminus.
So, as long as the putative operator sequence is known, µMACS MicroBead purification facilitates the isolation of uncharacterized DNA binding proteins

25. Translation control studies with µMACSTM Streptavidin Kit
Purification of RNA binding proteins
RNA probe
MACS MicroBead
Complimentary 3' biotinylated oligo
RNA binding proteins can control the translation by regulating the half life of the RNA (stabilizer - destabilizer) and thereby the availability of the mRNA for the translation process.
A possible probe for the isolation of proteins which bind to a specific RNA can look like this: the specific RNA probe, hybridized to a complementary, biotinylated oligonucleotide, bound by µMACS Streptavidin MicroBeads.
RNA / oligo / µMACS MicroBead complex
The probe for isolation of RNA binding proteins was generated by a full length transcript of the RNA, hybridized to a specific 3' biotinylated ss-oligonucleotid bound to µMACS Streptavidin MicroBeads.

26. Translation control studies with µMACSTM Streptavidin Kit
Purification of RNA binding proteins
Full length RNA column Mutant RNA column
Silver Staining of SDS-PAGE. Yeast crude extract was precleared with heparin agarose and subsequently incubated with the RNA/mutant RNA-µMACS Streptavidin MicroBead probe.
Yeast crude extract was precleared with heparin agarose and subsequently incubated with a specific full length RNA bound to a 3´biotinylated complimentary ss-oligo and magnetically labeled with µMACS Streptavidin MicroBeads. As a control a full length RNA with mutated binding side was used.
The silver stained SDS-PAGE shows flow through, several washes and the eluate. 4 proteins can be isolated, which bind specifically to the RNA sequence.
There is a lot of unbound protein in the flow through of the column. The stringency can easily be increased with further washing steps so that more and more unspecific bound proteins are removed and the eluate contains the specifically bound proteins only.
In the control experiment with the mutated RNA sequence these specific proteins cannot be isolated.
(Courtesy of Dr. Allan Albig, Washington State University, USA)

27. µMACS in proteomics research
• Protein isolation
• Interaction partner isolation
• Protein complex isolation
• Enzymatic, on-column reactions with the temperature-controlled thermoMACS Separator
Today, more and more research is directing at protein analysis. The relative amount of proteins, post-translational modifications and phosphorylation as well as the interaction of different proteins is crucially important.
µMACS MicroBeads are used for the isolation and further characterization of proteins or its binding partners.
Proteins or interacting proteins cannot only be isolated, they can also be used for enzymatic reactions directly on the MACS Column: For incubations at 37 or 42°C the thermoMACS, a heatable MACS Separation Unit can be used. By this e.g. kinase assays, enzymatic restrictions and many other reactions can be performed. The enzyme or the substrate are simply added, and after incubation the easy washing steps allow purification of the processed protein or the elution of the modified substrate for further analysis e.g. on PAGE gel.