Western Blot Protocol

2015-03-02

azim58 - Western Blot Protocol

See also
-[cni_Western Blot Protocol at CNIO 03-01-2015d1500]
-[cni_cell lysis protocol at cnio 03-01-2015d1502]

Western Blot Protocol
Here's the original protocol I received: Western Blot Protocols.pdf
Here's some Western Blot Images

Introduction:
A Western Blot is an analytical technique that is used to detect specific
proteins. It is a process that starts with SDS/Page gel electrophoresis
which separates denatured proteins by their size. Once separated, the
proteins are transferred to a nitrocellulose membrane where they are
probed using antibodies specific to the protein of interest.

Equipment:
Pipettes
Heating block
Centrifuge
XCell4 SureLock™ Midi-Cell Runner (Invitrogen, Cat# WR0100)
Mic;li-Trans Blot Electrophoretic Transfer Cell (Bio-Rad, Cat # 170-3930)
Power Supply (Bio-Rad, Cat # 164-5050)
Incubator, 5% CO2, 3rC

Materials:
Ladder/Marker

* See Blue Plus 2 Pre-stained standard (Invitrogen, Cat# LC5925)
* Precision Plus Protein Dual Color Standard from Biorad
  o Last seen in yellow box in door of -20 freezer by autoclave

SDS 4x Loading dye (BioRad, Cat# NP0007)
2-mercapotethanol (Sigma, Cat# M7154)
4-12% Bis-Tris NuPAGE Novex Precast Gel (Invitrogen, Cat # WG1403BOX)
1x Tris/Glycine/SDS Electrophoresis Buffer, dilute from 10X (Bio-Rad Cat#
161-0772EOU)
1x Transfer Buffer-
100mL 10X Transfer Buffer (Glycine, Tris Base), 200mL Methanol, up to
1000mL with molecular grade
water. or
1 L 1 x Transfer buffer recipe:
14.4 g
3.02 g
20%
GlYCine Electrophoresis Grade (MP Biomedicals, Cat # ICN808822)
Tris Base Electrophoresis Grade (Fisher Scientific, Cat # BP152-500)
Methanol (Fisher Scientific, Cat # A412-1)
Blotting Sandwich (Bio-Rad, Cat# 162-0235)
Vacutainer (BO, Cat # 8304250)

Procedures:
Polyacrylamide Gel

* Prepare samples for gel by suspending them in loading reagent.
  Samples are run alongside a ladder (often 4 uL).
  o Running buffer recipe per sample: 2.5 uL loading reagent (80%
    dye; 20% mercaptoethanol reducing agent), 1 uL Sample, 7.5 uL 1 x MES
    Buffer, 10 uL total volume.
    - different volumes can be used as well of course
    - The MES is not necessary. The sample can be diluted directly
      with 4X loading reagent.
    - A western blot can detect about 100 ng protein or less

* Prior to loading the gel, heat samples for 10 min at 90°C in dry
  heat bath. Spin down vial prior to opening and loading gel.
* Assemble SDS page gel cassette. Make sure to remove white tape strip
  at bottom of gel. Gel wells should be facing in. Load empty block into
  back slot if two gels are not being run.
* Load samples into the gel suspended in 1x MES buffer. Add 1X MES
  buffer so that buffer fills up the inside of the compartment and
  probably about half-way up the outside of the compartment. Load the
  ladder as well (4 uL)
* You may want to load duplicate samples (one for a coomassie stain or
  silver stain (a coomassie stain or silver stain (more sensitive) will
  detect all of the proteins present, whereas the antibody probing of a
  Western will detect specific proteins) and one for the nitrocellulose
  membrane)
* Run gel at 160V for 60 min
* Transfer
  o Samples are transferred to a nitrocellulose membrane via a wet
    transfer method.
  o Use ice cold transfer buffer. Transfer buffer can be made and
    reused up to 3x before discarding.
  o Assembly of the Transfer Cassette-
  o Remove the gel from the precast assembly and suspend in a tray of
    transfer buffer.
  o Handling Notes: All 'pre-wet" components are pre-wet with ice
    cold transfer buffer. Ensure to keep all components wet during
    assembly, do not allow drying to occur.
  o Cassette Assembly-BioRad

Make sure to roll out bubbles.
Black Electrode-
Black Cassette Back
Pre-wet Sponge
Pre-wet filter paper (-4.0mm)
Polyacrylamide gel
Pre-wet nitrocellulose membrane with shiny side touching gel
(alternatively PVDF membrane can be used but must be soaked in methanol.
Even then though the nitrocellulose is better from what I understand)
(Using wet filter paper above NC membrane roll out bubbles and then
continue with assembly)
Pre-wet filter paper (-4.0mm)
Pre-wet Sponge
Clear. Cassette front
Red Electrode

o Insert assembled transfer cassette into the rig with ice block and
  fill with transfer buffer to the top of the cassette.
o Run at 60V for 60 min
o During transfer, process can be checked by unplugging the transfer
  rig and opening the cassette and pealing the corner of the filter paper
  back to view the membrane. If the transfer requires more time, the rig
  can be reassembled and plugged back in to continue for additional 5-10
  min increments.
o Placing the transfer rig in an ice bucket with crushed ice will
  further ensure it does not overheat.
o You may want to do a coomassie stain on one of the gels or part of a
  gel to detect all of the proteins present, whereas the antibody probing
  will detect specific proteins.
*

* Blocking-
  o Remove the membrane from the assembly with forceps and transfer
    directly into 1 % BSA in TBST.. You may want to cut the bottom right
    corner of front to keep track of the orientation.
  o Incubate at 4° C overnight or 30 min - 2 hr room temperature.

* Primary Antibody
  o Antibody is diluted 1 :1,000 in 5% BSA in TBST (often 20 uL in 20
    mL)
  o Membrane is incubated in Primary Antibody Solution for 1-2 hrs at
    room temp while rotating
  o Wash 3X with TBST (may want to wash on rocker for 5 min intervals)

* Secondary Antibody
  o Dilute biotinylated secondary antibody 1:10,000 in 5% BSA in TBST
    (often 2 uL in 20 mL)
  o Incubate membrane with antibody for 1 hour
  o Wash 3X with TBST (may want to wash on rocker for 5 min intervals)

* Tertiary
  o Dilute streptavidin dye 1:10,000 in 5% BSA in TBST (often 2 uL in
    20 mL)
  o Incubate membrane with antibody for 1 hour in the dark
  o Wash 3X with TBST (may want to wash on rocker for 5 min intervals)
  o Dry membrane (it can be dried faster by pressing between two dry
    sponges; may want to dry for 10-20 min)

* Image with Typhoon
  o Turn on the computer for the Typhoon and select the Typhoon
    Scanner icon.
  o Set to fluorescence
  o Click Setup
  o Deselect laser options
  o Select the appropriate Emission and excitation lasers
  o Last Settings used were: Excitation: 633 nm, Emission filter: 670
    nm, Dye: AF647
  o May want to reduce the PMT to 350
  o Orient membrane on scanner
  o Select blocks to scan and click "scan"
  o Ensure to save file on server or flash drive instead of on the
    computer or the file will be deleted later. Note that the standard
    .gel files do not open well in other programs so make sure to at
    least save a .tif file.

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