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)
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
General features of the MicroBeads
• Super-paramagnetic
• Colloidal non-sedimenting
• Extremely small
• Biodegradable & non-toxic
Extremly fast reaction
kinetics & efficient
magnetic labeling
Magnetic protein isolation
1. Cell lysis
2. 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
Immunoprecipitation with µMACSTM Protein A/
Protein G MicroBeads
Pure protein in less than 2 hours
Cell lysis and
immunolabeling
Magnetic
separation
SDS-PAGE or
enzymatic assay
Immunoprecipitation with µMACS
IP of SV40 large T antigen from COS cells with
µMACS Protein G MicroBeads
TM
Silver stained SDS PAGE
L: Lysate
M: Marker
Lane 1: Large T antigen
(indicated by arrow)
Lane 2: Isotype control
Lane 3: Negative control
Immunoprecipitation of surface antigens
IP of biotinylated surface antigens of B cell line
JOK-1 with µMACS Protein A
TM
1
Western Blot with Streptavidin-HRP
2
3
4
CD 22 (140 kDa)
Lane 2: anti-CD22 (HD 239)
Lane 4: anti-MHC class I (W6/32)
Lane 1 and 3: isotype matched antibody as
control.
MHC Class I (45 kDa)
Co-IP of the androgen receptor and it´s target
Western Blot of Immunoprecipitated AR
using Anti-AR antibody
Western Blot of AR-coimmunoprecipitated Beta-Catenin using
anti-Beta-Catenin antibody
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
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). AntiAR antibody (lane 1, 3) or as a control anti-IgG (lane 2,
4) were used for Chip.
Courtesy of D. Mulholland, Vancouver, Canada
µMACS Epitope Tag Protein Isolation Kits
TM
Application
isolation of recombinant proteins with epitope tags
and their interaction partners from eukaryotic cells
µMACS Epitope Tag Protein Isolation Kits
TM
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
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
MACS in Molecular Biology
GFP = Green Fluorescent Protein from Aequoria
victoria
Zur Anzeige wird der QuickTime™
Dekompressor “GIF”
benötigt.
BM 10/2002
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
µMACS™ Epitope Tagged Protein Isolation Kits
Standardized isolation of different proteins
• recombinant proteins A-C
isolated with protein specific
Ab
Zur Anzeige wird der QuickTime™
Dekompressor “GIF”
benötigt.
• tagged recombinant
proteins A-C isolated with
tag specific Ab
BM 10/2002
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
µMACS Epitope Tag Protein Isolation Kits
TM
Working scheme for isolation of tagged proteins
Lyse cells and
add MicroBeads
Apply to MACS
Column
Remove unbound
material by stringent
washing
Elute target protein
while MicroBeads
remain on column
Protein isolation with µMACS Anti-c-myc MicroBeads
TM
Isolation of c-myc-BDCA2 from biotinylated cell lysate
1 2 3 4 5 6 7
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)
40 kDa 30 kDa -
24 kDa -
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)
Isolation c-myc-BDCA-2 using anti-HA and
anti-c-myc MicroBeads
anti-c-myc
anti-HA
35S-methionine
labeled isolation
c-mycBDCA-2
© Miltenyi Biotec
µMACS Streptavidin Kit
TM
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
µMACS Strepavidin Kit
TM
Isolation of DNA-binding proteins
1
2
3
4
5
Isolation of target molecules with biotinylated probes, here the isolation of DNAbinding 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).
6
Transcription control studies with µMACS
Streptavidin Kit
TM
Purification of DNA binding proteins
Marker
IL-4
non
stimulated stimulated
 Stat6
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 SDSPAGE and Western blotting
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
Isolation of a transcription factor
from E. coli expressing the recombinant factor
M
1
2
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
Translation control studies with µMACS
Streptavidin Kit
Purification of RNA binding proteins
RNA probe
MA CS MicroBead
Complimentary 3' biotinylated oligo
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.
TM
Translation control studies with µMACS
Streptavidin Kit
TM
Purification of RNA binding proteins
Full length RNA column
Mutant RNA column
Silver Staining of SDSPAGE. Yeast crude
extract was precleared
with heparin agarose
and subsequently
incubated with the
RNA/mutant RNAµMACS Streptavidin
MicroBead probe.
µMACS in proteomics research
• Protein isolation
• Interaction partner isolation
• Protein complex isolation
• Enzymatic, on-column reactions
with the temperature-controlled
thermoMACS Separator