Magnetic bead isolation of Mitochondria and following western blot analysis

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The protein extracts and of the positive fraction can be frozen at -20°C.  
The protein extracts and of the positive fraction can be frozen at -20°C.  
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Revision as of 15:50, 13 October 2009

Contents

General Information and authors


Short name/eponym:Magnetic bead isolation of mitochondria

Version:Version 1.0

Date of submission:27-October-2008


Responsible author:Margot Grandl

Institute/Department:Institute for Clinical Chemistry and Laboratory Medicine

University Hospital of Regensburg

Franz-Josef-Strauss-Allee 11

D-93053 Regensburg

Germany


Purpose


In focus is the isolation of mitochondria via Anti-TOMM22 MicroBeads from adipocytes after in vitro adipogenic differentiation, from THP-1 cell and primary monocytes.


Short explanation of the principle


TOMM22 is mitochondrial outer membrane protein. Magnetic Beads (MACS-beads) were coupled to an antibody against TOMM22 to isolate mitochondria via magnetic columns (Milteniy Biotech).


Materials required for the isolation procedure


PEB-buffer:PBS, steril

2mM EDTA

0.5% BSA

Proteaseinhibitor: Complete Mini EDTA-free

(Roche; Cat#: 04 693 159 001)

PEB+ Proteaseinhibitor:1 Mini EDTA-free complete tablet in 10ml PEB

Beads:Anti-Tomm22.MicroBeads (Miltenyi Biotech)

Columns:LS-Columns (Miltenyi Biotech; Cat #: 130-042-401)

Magnet:QuadroMACS-magnet (Miltenyi Biotech)

(QuadroMACS Starting Kit; Cat#: 130-091-051)


Materials required for western blot

RIPA-buffer:25 mM Tris•HCl pH 7.6

150 mM NaCl

1% NP-40

1% sodium deoxycholate

0.1% SDS

(PIERCE, Cat#: 89900 89901)

BC AssayProtein detection kit (Uptima Interchim; Cat#: UP40840A)

BSA2mg/ml solution in steril H2O

Gele4-12 % Bis-Tris NuPAGE® Gel

(Invitrogen; Cat#: NP0321-2)

Gel Cell Invitrogen XCell SureLockâ„¢ Mini-Cell

(Invitrogen; Cat#: EI0002)

WB-Running Buffer: 20xNuPAGE® MOPS SDS Running Buffer

(Invitrogen, Cat#: NP0001-02)

to use: 760 mL H2O (millipore) + 40 mL MOPS-Puffer

Sample-Buffer:100 µL DTT

250 µL NuPAGE® LDS Sample Buffer (4X) (Invitrogen; Cat#: NP0007)

650 µL H2O (millipore)

10 min at 95 °C

Membrane PVDF Membrane (0.2µm)

Transfer Buffer:20% Methanol

25mM Tris-HCl

192mM Glycin

add 1000 ml H2O; store at 4°C

Antibodies:

mitochondrial specific markers:cytochrom C (abcam; Cat#: ab13575)

Hsp60 (abcam; Cat#: ab46798)

Lipid droplet specific markers:perilipin (Progene Biotechnik; Cat#: GP33)

adipophilin (Progene Biotechnik; Cat#:GP41)

TIP74 (Progene Biotechnik; Cat#: GP32)

Detection reagent:ECL Plus western blotting detection system

(GE Healthcare; Cat#: RPN2132)


Sample, biological material(s) required for the procedure

  • Minimal amount of material: 1x105 cells/ml
  • Optimal amount of material: 1x107 cells/ml
  • Protein extract:10 - 20µg / analysis


Isolation procedure


  • Adipogenic differentiation of human primary preadipocytes for max. 25 days. THP-1 cells are suspension cell line, human monocytes can be isolated via magnetic beads (SOP: Monocytes isolation with magnetic beads)
  • After cell culture experiments take min.1x105 cells and max. 1x107 cells in 1ml PBS with protein inhibitors (sterile) and make a cell-lysate by pulling it about 15 times to a 26g needle
  • Cell lyses can be checked by using trypan blue reagent. The intact cells can exclude the reagent. The contingent of these intact cells can be detected by cell counting in “Neubauer chamber”.

Give 10µl of trypan blue reagent to 200µl of cell-lysate, after 5min incubation check the cell lyses. The efficiency should be 60-70%.

  • In the next step give 25µl anti-TOMM22 MicroBeads to 10ml PEB-buffer with protease inhibitor
  • Fulfill 1ml cell-lysate (1x105 - 1x107 cells) to 10ml anti-TOMM22 in PEB + protease-inhibitor

This approach can be up-scaled:


cells in PBS
with protein inhibitor
PEB [ml]
Anti-TOMM22.Microbeads [µl]
1*107/ml
10
25
2*107/ml
20
50
3*107/ml
30
75
4*107/ml
40
100
5*107/ml
50
125
  • Incubate 1h at 4°C
  • Place the LS-Column into the QuadroMACS-magnet and equilibrate each LS-Column with 3ml PEB-buffer
  • Give maximal 10ml sample (=1x107 cell-lysate) onto one column, 3 x 3,33ml samples onto the column and collect the flow-through
  • Wash the column with 3ml PEB three times and pool it with the flow-through, you get the negative fraction
  • Elute the positive fraction with the stamp without the magnet. This is the positive fraction
  • If you want to concentrate the sample, the positive fraction can be centrifuged at 13000xg, 2min, 4°C and subsequently diluted in the desired volume

Analysis of isolated cell lysates with western blot


Gel-Run:

  • Mix protein extract with sample buffer in order to get a protein concentration of 1µg/µl.
  • Load 10 – 20 µg per gel line
  • Use as standard rainbow marker (BioRad), 10µl/lane
  • gel-run at 200 V in Invitrogen XCell SureLockâ„¢ Mini-Cell for 45-50min.

Blotting:

  • Put the gel into the transfer buffer for 10min
  • Humidify the PVDF membrane in 100% methanol for 1-2min, transfer the membrane into the reservoir with transfer buffer and let it there for additional 5-10min
  • Assemble a “blot sandwich”: blottin pad (1-2), whatman paper, gel, membrane, whatman paper, blotting pads (1-2)
  • Blotting at 200V (350 mA) in Invitrogen XCell SureLockâ„¢ Mini-Cell for 1.5-2h

Protein detection with specific antibody:

  • After blotting wash the membrane with PBS + 0.1%Tween 20 buffer, 2-3 times
  • Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive), with a minute percentage of detergent such as Tween 20.

Blocking for 1h (usage of BSA or non-fat dry milk depends on antibody, check the datasheet of the antibody you use)

  • Antibody treatment: the dilution is specific for each antibody; also here check the datasheet from the provider.

Detection:

Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody.

The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. So-called "enhanced chemiluminescent" (ECL) detection is considered to be among the most sensitive detection methods for blotting analysis.

  • After treatment with secondary antibody membrane will be washed in PBS-0.1% Twenn 20 buffer for 2-3 times.
  • For detection the information of the manufacturer shall be used.

Analysis and Expected results

The mitochondria positive fraction should be checked for contaminations of the MAM (mitochondrial associated membrane) compartment using Western Blot. The contaminations could be peroxisomes, smooth ER and lipid droplets.


Storage and banking of residual materials

The protein extracts and of the positive fraction can be frozen at -20°C.

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