Electrophoresis + Blotting + Imaging

12354
Stain-Free Western Blotting
Faster Results. Better Data.

Traditional Workflow
Total Time: 16 hr
StepWorkflow

TimeData

1Gel Preparation

>1 hr

—

2Electrophoresis

~1 hr

—

3Transfer

1–3 hr—4Antibody
Incubation

~5 hr

—

5Imaging andAnalysis>30 min
Target proteins

6Strip and Reprobe
Often need reprobing for actin/tubulin as loading control~5 hr
Loading control

Stain-Free Western Blotting
Total Time: 5 hr
StepWorkflowTimeSeparateProteins~15min

DataProtein separation at 300 V

Visualize
Separation1 min
Stain-Free image ofpretransferred gel

TransferProteins

3–7minAssessTransfer

2 min—Stain-Free image of blot

Analyze
Western Blot
by Total Protein
Normalization~4.5 hrChemiluminescence detection

Fluorescence detection

Stain-Free Western Blotting
Separate Proteins
Rapidly separate proteins inas little as 15 minutes withCriterion and Mini-PROTEANTGX Stain-Free Precast Gels.
TGX chemistry offers superior protein separation
with fast run times, features a one-year shelf life,and uses standard Tris-glycine running buffers.

Protein separation at 300 V

Stain-Free technology is a sensitive, time-saving
alternative to traditional Coomassie staining
and is compatible with western blotting. No
staining or destaining is required, offering a
streamlined workflow.
Mini gel typical run times
Protein separation at 200 V
Mini handcast Tris-HCl gel (200 V)
Mini-PROTEAN TGX Stain-Free Gel (200 V)
Mini-PROTEAN TGX Stain-Free Gel (300 V)

Midi gel typical run times
Midi handcast Tris-HCl gel (200 V)
Criterion TGX Stain-Free Gel (200 V)
Criterion TGX Stain-Free Gel (300 V)
010203040Time, min506070Fig. 1. Protein separation at 300 V or 200 V yields similar results.
Any kD Criterion TGX Stain-Free Precast Gels run for 30 min (300 V)
and 45 min (200 V).

Faster Results. Better Data.
Visualize Separation
Immediately visualize separation
with Stain-Free technology andthe ChemiDoc MP Imaging System
in one easy step.
Advantages:■■■

Stain-Free gel activation and imaging (1 min)

Ensure all proteins are separated
 ave time staining proteins (1 min vs. 2 hrSfor Coomassie staining) un just 1 gel (Coomassie staining requiresRa separate gel)

Coomassie-stained gel (overnight staining)

Fig. 2. Results using a Stain-Free gel are similar to thoseusing a Coomassie-stained gel. Any kD Criterion TGX Stain-Free
Precast Gel run at 200 V for 45 min. Stain-Free technology isvisualized on the ChemiDoc MP Imaging System and compared toa gel stained overnight with a Bio-Safe Coomassie Stain.

Transfer Proteins
The Trans-Blot Turbo Transfer
System enables rapid and efficienttransfer of proteins across a wide
range of molecular weights.
The Trans-Blot Turbo System offers rapid
protein transfer efficiency — achieved in as littleas 3 minutes — compared to tank blotting.
Trans-Blot Turbo System (7 min)Semi-dry blotting (30 min)

Tank blotting (60 min)Fig. 3. Transfer efficiencies are comparable between the Trans-BlotTurbo System and tank blotting. Transfers were performed with theTrans-Blot Turbo System (7 min), the Trans-Blot SD Semi-Dry Transfer
Cell (30 min), and by tank transfer (60 min).

Assess Transfer
Verify high-quality transfer using
Stain-Free technology by instantly
imaging the membrane on theChemiDoc MP Imaging System.
Advantages:■

Check transfer efficiency■

Detect air bubbles and uneven transfers■No need for separate Ponceau S stainingStain-Free technology

Ponceau S Stain

Fig. 4. Stain-Free technology has a higher protein staining
efficiency compared to the Ponceau S Stain. Visualization of
the previously activated Stain-Free technology for total protein on ablot compared with a membrane stained with the Ponceau S Stain
for 1–2 min.

Analyze Western Blot byTotal Protein Normalization
Perform multiplex or
chemiluminescent blot detection
and total protein normalization
using the ChemiDoc MP Imaging
System and Image Lab Software.
The ideal western blot normalization signal:■■■ rovides 1:1 change in signal with change
Pin total protein loadedIs linear over the widest range of total
protein loaded oes not depend on the expression of aD
single protein being constant for all
experimental treatments

Total protein normalization is recommended
because it satisfies all three characteristics.
Housekeeping protein (HKP) normalization isnot recommended because the signal:■

 epends on the expression of a single protein
Dbeing constant for all experimental treatments
 ends to be linear only at a low amount of total
Tprotein load (up to ~10 µg/lane)

A. Stain-Free blot image
5040302010B. b-actin
5040

E. Stain-Free total protein vs. housekeeping proteins
3020C. b-tubulin
5040302010D. GAPDH
5040302061010Relative intensity of protein bands■Stain-FreeActinGAPDHTubulin
Quantitative response

5432
100

10203040HeLa cell lysate, µg50Fig. 5. Total protein detection using Stain-Free technology provides a 1:1 signal relative to protein load over a wide range of total protein load.
A, Stain-Free total protein signal. B, b-actin signal. C, b-tubulin signal. D, GAPDH signal. E, comparison of linearity of total protein normalization
vs. housekeeping proteins over a range of protein concentrations.

60Ordering Information
Mini-PROTEAN TGX Stain-Free Precast Gels

10-Well
30 μl

Description

10-Well
50 μl

12-Well
20 μl

15-Well
15 μl

IPG Well
7 cm IPG Strip

7.5% Resolving Gel

45680234568024456802545680264568021

10% Resolving Gel

45680334568034456803545680364568031

12% Resolving Gel

4568043456804445680454568046

4568041

4–15% Resolving Gel

45680834568084456808545680864568081

4–20% Resolving Gel

45680934568094456809545680964568091

8–16% Resolving Gel

45681034568104

45681054568106

4568101

Any kD Resolving Gel

45681234568124456812545681264568121

18-Well
30 μl26-Well
15 μl

Prep+2-Well*
700 μl

IPG+1-Well*
11 cm IPG Strip

Criterion TGX Stain-Free Precast Gels**

12+2-Well*
45 μl

Description
7.5% Gel

567802356780245678025——

10% Gel5678033

5678034

5678035——

12% Gel

5678043

56780445678045——

4–15% Gel

5678083

5678084

5678085

56780825678081

4–20% Gel

567809356780945678095

56780925678091

8–16% Gel

56781035678104

567810556781025678101

Any kD Gel

56781235678124567812556781225678121* Reference wells accommodate 15 µl of markers/standards. ** Criterion TGX Stain-Free Gels are sold singly.
Catalog #Description

Imaging Systems
12003154	ChemiDoc MP Imaging System, blot and gel imaging
system, UV/visible light imaging, chemiluminescence,
5 fluorescence channels (RGB, far red, near-IR).
Includes internal computer, 12" touch screen display,
Image Lab Touch Software, blot/UV/Stain-Free
sample tray
12003153	ChemiDoc Imaging System, blot and gel imaging
system, UV/visible light imaging, chemiluminescence,
upgradeable for multiplex fluorescence detection.
Includes internal computer, 12" touch screen
display, Image Lab Touch Software,
blot/UV/Stain-Free sample tray
Protein Standards
1610373	Precision Plus Protein All Blue Standards
1610363	Precision Plus Protein Unstained Standards
1610385	Precision Plus Protein WesternC Pack
Buffers1610732		
10x Tris/Glycine/SDS
1610747
4x L
 aemmli Sample Buffer
Electrophoresis Cells
1656001	
Criterion Cell, includes electrophoresis buffer tank,lid with power cables, 3 sample loading guides
1658004	
Mini-PROTEAN Tetra Cell for Mini Precast Gels, 		
4-gel vertical electrophoresis system, includes 		
electrode assembly, companion running module,tank, lid with power cables, mini cell buffer dam

Catalog #Description

Power Supplies
1645050
1645070

PowerPac Basic Power Supply
PowerPac Universal Power Supply

Blotting Materials
1704150
Trans-Blot Turbo Transfer System, blotting
instrument, includes base, 2 cassettes to hold 1–2 midi
or up to 4 mini blotting sandwiches, blot roller
1704156	Trans-Blot Transfer Pack, mini, PVDF, pkg of 101704157	
Trans-Blot Transfer Pack, midi, PVDF, pkg of 101704158	Trans-Blot Transfer Pack, mini, nitrocellulose, pkg of 101704159	Trans-Blot Transfer Pack, midi, nitrocellulose, pkg of 101704270	Trans-Blot Turbo RTA Transfer Kit, mini, nitrocellulose
1704271	Trans-Blot Turbo RTA Transfer Kit, midi, nitrocellulose
1704272	Trans-Blot Turbo RTA Transfer Kit, mini, PVDF
1704273	Trans-Blot Turbo RTA Transfer Kit, midi, PVDF
1704274	Trans-Blot Turbo RTA Transfer Kit, mini, LF PVDF
1704275	Trans-Blot Turbo RTA Transfer Kit, midi, LF PVDF

Visit bio-rad.com/StainFreeWB for more information.
BIO-RAD, MINI-PROTEAN, and TRANS-BLOT are trademarks of
Bio-Rad Laboratories, Inc. in certain jurisdictions. TGX Stain-Free
Precast Gels are covered by U.S. Patent Numbers 7,569,130 and8,007,646. All trademarks used herein are the property of theirrespective owner.

Bio-RadLaboratories, Inc.
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