Novex® Tris-Glycine Gels - Invitrogen
Novex® Tris-Glycine Gels - Invitrogen
Novex® Tris-Glycine Gels - Invitrogen
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<strong>Novex®</strong> <strong>Tris</strong>-<strong>Glycine</strong> <strong>Gels</strong><br />
TABLE OF CONTENTS<br />
PRODUCT DESCRIPTION<br />
SHIPPING CONDITIONS<br />
STORAGE CONDITIONS<br />
STABILITY<br />
QC SPECIFICATIONS<br />
PROTOCOL & APPLICATION NOTES<br />
Procedure Overview<br />
Denaturing Conditions<br />
Denaturing Sample Preparation<br />
Denaturing <strong>Tris</strong>-<strong>Glycine</strong> Buffer Preparation<br />
Non-Denaturing (Native) Conditions<br />
Native Sample Preparation<br />
Native <strong>Tris</strong>-<strong>Glycine</strong> Buffer Preparation<br />
Reducing Agent<br />
Electrophoresis Protocol<br />
Buffer Recipes<br />
Staining<br />
Gel Running Anomalies<br />
Other Applications<br />
ALTERNATE PRODUCTS & COMPATIBILITY<br />
PRODUCT DOCUMENTATION<br />
REFERENCES<br />
PRODUCT NAME & CATALOG NUMBER<br />
COMPONENTS<br />
ASSOCIATED PRODUCTS<br />
RELATED TECHNICAL SUPPORT NOTES<br />
1
PRODUCT DESCRIPTION<br />
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<strong>Novex®</strong> <strong>Tris</strong>-<strong>Glycine</strong> polyacrylamide gel chemistry is based on the Laemmli system with minor modifications for maximum<br />
performance in the pre-cast format. These gels do not contain SDS and can therefore be used to accurately separate both<br />
native and denatured proteins. <strong>Novex®</strong> <strong>Tris</strong>-<strong>Glycine</strong> <strong>Gels</strong> provide reproducible separation of a wide range of proteins into<br />
well resolved bands.<br />
Gel Matrix: Acrylamide/Bis-Acrylamide<br />
Gel Thickness: 1.0mm and 1.5mm<br />
Gel Size: 8cm X 8cm (H X W)<br />
Cassette Size: 10cm X 10cm (H X W)<br />
Formulation: <strong>Tris</strong>-<strong>Glycine</strong> gels are made with <strong>Tris</strong> base, HCl, acrylamide, bisacrylamide, TEMED, APS, and ultra pure<br />
water.<br />
SHIPPING CONDITIONS<br />
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<strong>Gels</strong> are shipped on wet ice.<br />
STORAGE CONDITIONS<br />
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<strong>Gels</strong> and buffers should be stored at +4 degrees centigrade.<br />
STABILITY<br />
(back to Table of Content)<br />
Shelf life varies from 4-8 weeks depending upon gel type.<br />
QC SPECIFICATIONS<br />
(back to Table of Content)<br />
A Certificate of Analysis available for each gel and buffer lot on our web site.<br />
PROTOCOL AND APPLICATION NOTES<br />
(back to Table of Content)<br />
Procedure Overview<br />
Gel Running Anomalies<br />
Other Applications<br />
Procedure Overview<br />
(back to Table of Content)<br />
(back to Protocol and Application Notes)<br />
Denaturing Conditions<br />
Electrophoresis is performed under denaturing conditions using an anionic detergent such as sodium dodecylsulfate (SDS).<br />
SDS denatures and unfolds the proteins by wrapping around the hydrophobic portions of the protein. SDS binds at a ratio of<br />
~1.4 g SDS per gram of protein. The resultant SDS-protein complexes are highly negatively charged and migrate through the<br />
gel based on their size rather than charge.<br />
Denaturing Sample Preparation<br />
Prepare sample by adding one part <strong>Novex®</strong> <strong>Tris</strong>-<strong>Glycine</strong> SDS Sample Buffer (2X) to one part sample and mix well.<br />
Heat the samples (reduced or non-reduced) at 85°C for 2-5 minutes for optimal results. Heating the sample at 100°C in<br />
SDS containing buffer results in proteolysis.<br />
2
Denaturing <strong>Tris</strong>-<strong>Glycine</strong> Buffer Preparation<br />
Dilute 10X <strong>Tris</strong>-<strong>Glycine</strong> Running Buffer as follows:<br />
<strong>Tris</strong>-<strong>Glycine</strong> Running Buffer (10X) 100ml<br />
Ultra pure Water 900ml<br />
Total volume: 1 liter<br />
Invert to mix<br />
Non-Denaturing (Native) Conditions<br />
Electrophoresis is performed under non-denaturing (native) conditions using buffer systems that maintain the native protein<br />
confirmation, subunit interaction, and biological activity. During native electrophoresis, proteins are separated based on their<br />
charge to mass ratios.<br />
Native Sample Preparation<br />
Prepare sample by adding one part <strong>Novex®</strong> <strong>Tris</strong>-<strong>Glycine</strong> Native Sample Buffer (2X) to one part sample and mix well.<br />
Do not heat native samples.<br />
Native <strong>Tris</strong>-<strong>Glycine</strong> Buffer Preparation<br />
Dilute <strong>Novex®</strong> <strong>Tris</strong>-<strong>Glycine</strong> Native Running Buffer (10X) 1:9 as follows:<br />
<strong>Novex®</strong> <strong>Tris</strong>-<strong>Glycine</strong> Native Running Buffer 100ml<br />
Ultra pure Water 900ml<br />
Total volume: 1 liter<br />
Invert to mix.<br />
Reducing Agent<br />
The NuPAGE® Reducing Agent contains 500 mM dithiothreitol (DTT) at a 10X concentration in a ready-to-use, stabilized<br />
liquid form and is used to prepare samples for reducing gel electrophoresis. β mercaptoethanol can be used with the <strong>Novex®</strong><br />
gels at a final concentration of 2.5%. Choice of the reducing agent is a matter of preference and either DTT or βmercaptoethanol<br />
can be used. Add the reducing agent to the sample within an hour of loading the gel.<br />
Avoid storing reduced samples for long periods even if they are frozen. This will result in the reoxidation of samples during<br />
storage and produce inconsistent results.<br />
Running reduced and non-reduced on the same gel: Running reduced and non-reduced samples on the same gel is not<br />
recommended. If it cannot be avoided, do not run reduced and non-reduced samples in adjacent lanes. The reducing agent<br />
may have a carry-over effect on the non-reduced samples if they are in close proximity.<br />
Maximum recommended protein load amount per lane: Load 30 - 50 micrograms of protein per gel for optimum results.<br />
Maximum load is 100 micrograms per gel.<br />
Electrophoresis Protocol<br />
1. Cut open the gel pouch with scissors and remove the <strong>Tris</strong>-<strong>Glycine</strong> gel from pouch.<br />
2. Rinse the gel cassette with D.I. water. Peel off the tape from the bottom of the cassette.<br />
3. In one smooth motion, gently pull the comb out of the cassette.<br />
4. Use a disposable pipette to rinse the sample wells with 1X running buffer. Invert the gel and shake to remove the<br />
buffer. Repeat two more times.<br />
5. Orient two gels in the XCell SureLock TM Mini-Cell (or XCell II) so that the notched “well” side of the cassette faces<br />
inwards toward the buffer core. Seat the gels on the bottom of the Mini-Cell and seal into place with the rear gel<br />
tension wedge.<br />
NOTE: If you are running only one gel, the square plastic buffer dam replaces the second gel cassette.<br />
6. Check for tightness of seal by filling the inner buffer chamber with a small amount of buffer. If a leak is detected<br />
from inner to outer chamber, recollect the buffer, reseal the chamber and refill. Once the seal is tight, proceed to fill<br />
the upper buffer chamber (inner) with running buffer so that the buffer level exceeds the level of the wells.<br />
7. Run the gel according to either of the following running conditions.<br />
Laemmli SDS:<br />
Voltage: 125V constant<br />
Approximated Current: Start: 30-40mA/gel<br />
End: 8-12mA/gel<br />
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Run Time: Approximately 90 minutes (This is dependent on the gel percentage). The run is complete when the<br />
Bromophenol blue tracking dye reaches the bottom of the gel.<br />
Native <strong>Tris</strong>-<strong>Glycine</strong>:<br />
Voltage: 125V constant<br />
Approximated Current: Start: 6-12mA/gel<br />
End: 3-6mA/gel<br />
Run Time: 1-12 hours<br />
<strong>Gels</strong> should be immediately fixed, stained or transferred as desired<br />
Buffer Recipes<br />
<strong>Tris</strong>-<strong>Glycine</strong> SDS Sample Buffer<br />
Catalog No. LC2676 (2x) 20ml<br />
0.5 M <strong>Tris</strong>-HCl, pH 6.8 2.5 ml<br />
Glycerol 2.0 ml<br />
10% (w/v) SDS 4.0 ml<br />
0.1% Bromophenol blue 0.5 ml<br />
Distilled water to 10.0 ml<br />
<strong>Tris</strong>-<strong>Glycine</strong> SDS Running Buffer<br />
Catalog No. LC2675 (10x) 500ml<br />
Catalog No. LC26754 (10x) 4X1L<br />
<strong>Tris</strong> Base 29 g<br />
<strong>Glycine</strong> 144 g<br />
SDS 10 g<br />
Distilled water to 1 liter<br />
NOTE: 1x running buffer should be ~pH 8.3. Do not use acid or base to adjust pH.<br />
<strong>Tris</strong>-<strong>Glycine</strong> Native Running Buffer<br />
Catalog No. LC2672 (10x) 500ml<br />
<strong>Tris</strong> Base 29 g<br />
<strong>Glycine</strong> 144 g<br />
Distilled water to 1 liter<br />
NOTE: 1x running buffer should be ~pH 8.3. Do not use acid or base to adjust pH.<br />
<strong>Tris</strong>-<strong>Glycine</strong> Native Sample Buffer<br />
Catalog No. (2x) LC2673 20ml<br />
0.5 M <strong>Tris</strong>-HCl, pH 8.6 4.0 ml<br />
0.1% Bromophenol blue 0.5 ml<br />
Glycerol 2.0 ml<br />
Distilled water to 10.0 ml<br />
<strong>Glycine</strong> Transfer Buffer<br />
Catalog No. LC3675 (25x) 500ml<br />
<strong>Tris</strong> Base 18.2g<br />
<strong>Glycine</strong> 90.0g<br />
Distilled Water 500 ml<br />
Staining<br />
The <strong>Novex®</strong> Pre-Cast <strong>Tris</strong>-<strong>Glycine</strong> <strong>Gels</strong> are compatible with most silver staining protocols. The SilverQuest TM Silver<br />
Staining Kit or the SilverXpress® Silver Staining Kit is recommended. The <strong>Tris</strong>-<strong>Glycine</strong> <strong>Gels</strong> are also compatible with any<br />
of the standard Coomassie® staining procedures. The SimplyBlue TM SafeStain and <strong>Novex®</strong> Colloidal Coomassie® Blue<br />
Staining Kit are recommended.<br />
4
Gel Running Anomalies<br />
Streaking forward or frowning of samples on a gel: Some potential causes are:<br />
re-oxidation of protein during run or<br />
protein has highly hydrophobic regions where protein can exclude SDS.<br />
Steps you can take to improve results:<br />
Reduce samples right before loading and add antioxidant to running buffer. Do not use samples that<br />
have been stored in reducing agent.<br />
Load sample with 2X sample buffer instead of 1X.<br />
Add SDS to upper chamber buffer, try 0.1%, 0.2%, 0.3%, and 0.4% (It is not recommended to go over<br />
this).<br />
Dumbbell or barbell shaped bands: Barbell shaped bands are a result of loading too large of a sample volume. When<br />
a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins<br />
and the sample moves through the stacking portion of the gel, the sample will incompletely stack causing a slight<br />
retardation of the portion of the sample that diffused to the sides of the wells. This effect may be intensified for<br />
larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel. To<br />
alleviate the problem, concentrate the protein and load a smaller volume. This gives a "thinner" starting zone.<br />
Spreading, compressed lanesThe lanes are all cast the same width by the gel comb. However, there can be situations<br />
where a sample lane will spread or compress. A heavily loaded lane will spread if loaded next to lanes with much<br />
less or no sample (which will be compressed).<br />
Bubbles seen during or after a gel run: When the gels are being made, the acrylamide is sparged with helium for a<br />
certain length of time to remove all of the nitrogen gas from the mix. If the acrylamide is not sparged sufficiently,<br />
bubbles can be produced during the electrophoresis run.<br />
“Smiling” bands in the outside lanes of the gel: "Smiling" bands sometimes indicates that the acrylamide in the gel<br />
may be breaking down leaving less of a matrix for the proteins to migrate. <strong>Gels</strong> should not be used beyond their<br />
expiration date.<br />
Faint shadow or "ghost" band below a normal and expected protein band: Ghost bands are usually attributed to a<br />
slight lifting or bubbling of the gel off from the cassette, which results in the trickling-down of some sample beyond<br />
its normal migration point. It then accumulates and appears as a second faint band.<br />
Gel lifting off the cassette can be caused by:<br />
Expired gels that are degrading.<br />
Improper storage of gels.<br />
Too much heat accumulating during the electrophoresis run due to excessive current.<br />
Insufficient polymerization of the polyacrylamide.<br />
Streaking of a sample on a protein gel: Streaking can be the result of<br />
sample overload,<br />
addition of reducing agent that is not fresh or<br />
the presence of membranes and other complexes or DNA in your sample.<br />
Bands appear to be getting narrower (or "funneling") as they progress down the gel: There may be too much βME<br />
(Beta-mercaptoethanol), sample buffer salts, or DTT in your samples. If the proteins are over-reduced, they can be<br />
negatively charged and actually repel each other across the lanes. The bands get narrower as they progress down the<br />
gel and the proteins repel each other.<br />
Skirting: Skirting is a phenomena that is seen under a variety of circumstances. The most common of these<br />
circumstances is when the <strong>Tris</strong>-<strong>Glycine</strong> gels age or too much sample has been loaded. Another example of skirting<br />
occurred with a pegylated protein that is sensitive to ammonium persulfate (APS).<br />
Other Applications<br />
<strong>Tris</strong>-<strong>Glycine</strong> gels for Blue Native applications: <strong>Tris</strong>-<strong>Glycine</strong> or the <strong>Tris</strong>-Acetate gels can be used for this purpose.<br />
This is based on the work by Schagger and von Jagow. Blue Native PAGE permits a high-resolution separation of<br />
multi-protein complexes under native conditions. It is a charge shift method in which the electrophoretic mobility of<br />
a multi-protein complex is determined by the negative charge of bound Coomassie dye and the size and shape of the<br />
complex. Coomassie does not act as a detergent and preserves the structure of multi-protein complexes. It was<br />
developed for the separation of membrane proteins from membrane or organelle fractions. More Coomassie can be<br />
used in the running and sample buffer for a membrane protein vs. a soluble protein. If there is a smear present<br />
below the band of interest, this would indicate that the subunits are dissociating during the course of the run. This<br />
can be remedied by running it in the cold. This should help keep the subunits together. Use the buffers<br />
recommended in the published protocols.<br />
5
Use of MOPS buffer to run a <strong>Tris</strong>-<strong>Glycine</strong> gel: It is not recommended, because MOPS is highly conductive and it<br />
may cause streaking on a <strong>Tris</strong>-<strong>Glycine</strong> gel.<br />
Instances where the pI of the protein is 8.3, i.e. equal to that of the running buffer: Two solutions: a) add 0.02%<br />
SDS into upper chamber; b) add 0.25% Coomassie G250 into the sample and 0.02% Coomassie G250 into the upper<br />
chamber. Note that neither solution will denature the protein. They only make the protein slightly charged.<br />
Method to sharpen the band resolution for proteins run under native conditions: To sharpen bands of hydrophobic<br />
proteins in the native system, try adding 0.1% sodium deoxycholate to the upper buffer tank as well as the sample<br />
buffer.<br />
PRODUCT DOCUMENTATION<br />
(back to Table of Content)<br />
Brochures Citations Cell lines<br />
COA FAQ Licensing<br />
Manuals MSDS Newsletters<br />
Vector Data<br />
REFERENCES<br />
(back to Table of Content)<br />
Laemmli, U. K. (1970) Nature 227: 680 – 685.<br />
PRODUCT NAME AND CATALOG NUMBERS<br />
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<strong>Tris</strong>-<strong>Glycine</strong> <strong>Gels</strong><br />
Percentage Width, well<br />
number<br />
Catalog number Percentage Width, well number Catalog number<br />
4% 1.0 mm, 10 well EC6055BOX 16% 1.0 mm, 10 well EC6495BOX<br />
1.0 mm, 12 well EC60552BOX 1.0 mm, 12 well EC64952BOX<br />
1.0 mm, 15 well EC60555BOX** 1.0 mm, 15 well EC64955BOX<br />
1.0 mm, 2D well EC6056BOX** 1.0 mm, 2D well EC6496BOX**<br />
1.5 mm, 10 well EC6058BOX 1.5 mm, 10 well EC6498BOX<br />
1.5 mm, 15 well EC60585BOX 1.5 mm, 15 well EC64985BOX<br />
1.5 mm, 2D well EC6059BOX** 1.5 mm, 2D well EC6499BOX**<br />
6% 1.0 mm, 10 well EC6065BOX 18% 1.0 mm, 10 well EC6505BOX<br />
1.0 mm, 12 well EC60652BOX 1.0 mm, 12 well EC65052BOX<br />
1.0 mm, 15 well EC60655BOX 1.0 mm, 15 well EC65055BOX<br />
1.0 mm, 2D well EC6066BOX** 1.0 mm, 2D well EC6506BOX<br />
1.5 mm, 10 well EC6068BOX 1.5 mm, 10 well EC6508BOX<br />
1.5 mm, 15 well EC60685BOX 1.5 mm, 15 well EC65085BOX<br />
1.5 mm, 2D well EC6069BOX** 1.5 mm, 2D well EC6509BOX**<br />
8% 1.0 mm, 5 well EC6014BOX 4-12% 1.0 mm, 10 well EC6035BOX<br />
1.0 mm, 10 well EC6015BOX 1.0 mm, 12 well EC60352BOX<br />
1.0 mm, 12 well EC60152BOX 1.0 mm, 15 well EC60355BOX<br />
1.0 mm, 15 well EC60155BOX 1.0 mm, 2D well EC6036BOX<br />
1.0 mm, 2D well EC6016BOX** 1.5 mm, 10 well EC6038BOX<br />
1.0 mm, 1 well EC6011BOX** 1.5 mm, 15 well EC60385BOX<br />
1.0 mm, 2 well EC6012BOX** 1.5 mm, 2D well EC6039BOX**<br />
1.5 mm, 10 well EC6018BOX 8-16% 1.0 mm, 1 well EC6041BOX**<br />
1.5 mm, 15 well EC60185BOX 1.0 mm, 2 well EC6042BOX**<br />
6
1.5 mm, 2D well EC6019BOX** 8-16% 1.0 mm, 5 well EC6044BOX**<br />
10% 1.0 mm, 1 well EC6071BOX** 1.0 mm, 10 well EC6045BOX<br />
1.0 mm, 5 well EC6074BOX 1.0 mm, 12 well EC60452BOX<br />
1.0 mm, 10 well EC6075BOX 1.0 mm, 15 well EC60455BOX<br />
10% 1.0 mm, 12 well EC60752BOX 8-16% 1.0 mm, 2D well EC6046BOX<br />
1.0 mm, 15 well EC60755BOX 1.5 mm, 10 well EC6048BOX<br />
1.5 mm, 10 well EC6076BOX 1.5 mm, 15 well EC60485BOX<br />
1.5 mm, 15 well EC6078BOX 1.5 mm, 2D well EC6049BOX<br />
1.5 mm, 2D well EC60785BOX 4-20% 1.0 mm, 1 well EC6021BOX<br />
12% 1.0 mm, 1 well EC6001BOX 1.0 mm, 2 well EC6022BOX**<br />
1.0 mm, 2 well EC6002BOX** 1.0 mm, 5 well EC6024BOX<br />
1.0 mm, 5 well EC6004BOX 1.0 mm, 9 well EC60249BOX**<br />
1.0 mm, 9 well EC60049BOX** 1.0 mm, 10 well EC6025BOX<br />
1.0 mm, 10 well EC6005BOX 1.0 mm, 12 well EC60252BOX<br />
1.0 mm, 12 well EC60052BOX 1.0 mm, 15 well EC60255BOX<br />
1.0 mm, 15 well EC60055BOX 1.0 mm, 2D well EC6026BOX<br />
1.0 mm, 2D well EC6006BOX 1.5 mm, 10 well EC6028BOX<br />
1.5 mm, 10 well EC6008BOX 1.5 mm, 15 well EC60285BOX<br />
1.5 mm, 15 well EC60085BOX 1.5 mm, 2D well EC6029BOX<br />
1.5 mm, 2D well EC6009BOX 10-20% 1.0 mm, 1 well EC6131BOX**<br />
14% 1.0 mm, 1 well EC6481BOX** 1.0 mm, 10 well EC6135BOX<br />
1.0 mm, 2 well EC6482BOX** 1.0 mm, 12 well EC61352BOX<br />
14% 1.0 mm, 5 well EC6484BOX 1.0 mm, 15 well EC61355BOX<br />
1.0 mm, 10 well EC6485BOX 1.0 mm, 2D well EC6136BOX<br />
1.0 mm, 12 well EC64852BOX 4-20%Zoom 1.0 mm IPG well EC60261BOX<br />
1.0 mm, 15 well EC64855BOX<br />
1.0 mm, 2D well EC6486BOX<br />
1.5 mm, 10 well EC6488BOX<br />
1.5 mm, 15 well EC 64885BOX<br />
1.5 mm, 2D well EC6489BOX**<br />
** = Stocking type A. Products we sell but do not maintain inventory of. They are made as orders come in.<br />
Buffers and Solutions<br />
Product Catalog number Quantity<br />
<strong>Tris</strong>-<strong>Glycine</strong> SDS Running Buffer (10x) LC2675 500 ml<br />
<strong>Tris</strong>-<strong>Glycine</strong> SDS Running Buffer (10x) LC26754 4X1L<br />
<strong>Tris</strong>-<strong>Glycine</strong> SDS Sample Buffer (2x) LC2676 20 ml<br />
<strong>Tris</strong>-<strong>Glycine</strong> Native Running Buffer (10x) LC2672 500 ml<br />
<strong>Tris</strong>-<strong>Glycine</strong> Native Sample Buffer (2x) LC2673 20 ml<br />
<strong>Tris</strong>-<strong>Glycine</strong> Transfer Buffer (25x) LC3675 500 ml<br />
NuPAGE® Sample Reducing Agent (0.5M DTT) (10x) NP0004 250µl<br />
NP0009 10 mL<br />
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7