Delaive E, Arnould T, Raes M, Renard P.

Laboratory of Biochemistry and Cell Biology (URBC), University of Namur (FUNDP), 61 rue de Bruxelles, 5000 Namur, Belgium.

Western blotting is widely used in protein analysis, classically using enhanced chemoluminescence for protein detection. Fluorescence-labelled secondary antibodies have emerged in recent years for detection of antigens, in order to improve the sensitivity and the linear range of detection. Here we show that the sensitivity can be further improved by an additional step in the detection procedure: the antigen is detected by successive incubations with a primary antibody, followed by a biotinylated secondary antibody and then a tertiary fluorescent conjugate. Using the detection of different antigens by CyDye-conjugated secondary antibodies in a two-step protocol as a reference, two tertiary fluorescent conjugates were evaluated: CyDye-conjugated streptavidin and CyDye-conjugated anti-biotin antibody. An four-fold increase in sensitivity was achieved with CyDye-conjugated streptavidin; numerous unspecific bands were also generated. CyDye-conjugated anti-biotin antibody did not generate any unspecific bands and led to a 30-fold increase in sensitivity, compared to detection with CyDye-conjugated secondary antibody.

J Immunol Methods. 2008 May 20;334(1-2):51-8. Epub 2008 Mar 10.

Technorati : Fluorescent conjugate, Three-step procedure, Western blot detection

GR Safe can:

• Save money for your lab
• Avoid DNA mutation caused by ethidium bromide and UV light.
• Offer a safer and better alternative to ethidium bromide and expensive SYBR brand stains
• Eliminate risks to yourself, the environment and your institution

Feedbacks from Customers:

• Thanks for the free sample, it works and we are buying more.
• Good for my teaching class (high school).
• Nice stain.
• Better than EB!

Description: GR Safe is a new, safe nucleic acid stain for detecting double-stranded DNA, single-stranded DNA, and RNA in agarose gel. It can be used for replacing mutagenic ethidium bromide (EB). GR Safe emits green fluorescence when bound to dsDNA and red fluorescence when bound to ssDNA or RNA. This new stain has two fluorescence excitation maxima when bound to nucleic acid, one centered at approximately 290 nm and one at approximately 490nm. GR Safe is as sensitive as EB, and much cheaper than SYBR Green, SYBR Gold and SYBR Safe, and you can use GR Safe just as the way you used EB.

Compared to EB which is a very strong mutagen, GR Safe caused very few mutations in the Ames test. In addition, GR Safe had a negative result in mouse marrow chromophilous erythrocyte micronucleus test and mouse spermary spermatocyte chromosomal aberration test.

• Available at 10,000X in H₂O for better safety –No more toxic and flammable organic solvent
• Room temperature storage for better convenience, stable at room temperature for years –No more freeze-and-thaw cycle!
• As sensitive as Ethidium Bromide and SYBR Safe –With confidence in your mind: it works! .
• Add GR Safe to warm agarose gel solution as you did with ethidium bromide before–No need to do lengthy post electrophoresis staining, save your valuable time.
• DNA bands can be viewed using either UV or safer Blue Light Transilluminator. If you use Blue Light transilluminator, you will not expose to dangerous UV light, so you will not get sun burn or skin cancer or damaged eyes.
• You can use digital camera to take gel photos: No need to use expensive gel documentation equipment or Polaroid Camera and films.
• Will not affect downstream experiments: compatible with all gel purification kits tested, will not inhibit ligation reaction etc.
• Compatible with Sodium Borate Electrophoresis Buffer: Run gel 2-3 times faster at higher voltage, resolve shaper bands in minutes, and less heat generation.
• Watch DNA migrate at your bench, in real-time without UV light (LED bluelight lamp is available from http://www.labsupplymall.com.)
• Cut out DNA bands for subclonning under safer blue light: No mutations caused by EB and UV light. (Blue Light transilluminator will be available from http://www.labsupplymall.com soon).

Sensitivity:

p>GR Safe vs EtBr

GR Safe	EtBr

Storage: Store at room temperature.

Disposal:

• Gel: Biosafety trash bag.
• TAE or Borate Buffer Solution: sink or consult a chemical safety officer at your institution.

Protocol:

1. Prepare 40 ml of agarose gel solution (concentration from 0.8~2.0%) with TAE or Borate Buffer in a 250 ml flask and mix it thoroughly. Place the flask in the microware, heat on high until the solution is completely clear and no small floating particles are visible (about 2~3 minutes).

2. After the gel solution cool to about 55 oC, add 2 µl of GR Safe to the solution. Swirl the flask gently to mix the solution and avoid forming bubbles.

3. Pour the gel solution into a gel tray until the comb teeth are immersed about 1/4~1/2 into the gel solution.

4. After the agarose gel has solidified you can perform electrophoresis using either 1X TAE or 1X Borate Buffer (Available from http://www.labsupplymall.com).

5. Detect the bands using UV or blue light transilluminator.

FAQ

1, What filter should I use for blue light transilluminator?

Amber filter. You can buy it from www.bhphotovideo.com, www.adorama.com, www.ritzcamera.com or www.wolfcamera.com

2, Where to buy blue light transilluminator?

www.invitrogen.com/safeimager, www.clarechemical.com/transilluminator.htm.

3, Where to buy blue light LED (torch) for monitoring gel and cutting DNA band from gel?

Ebay, Ebay Motors or other on-line stores

4, I got high background, what should I do?

Use less GR Safe, e.g., 1 ul/per 100 ml gel solution

5, Can I use UV transilluminator?

Yes. You can also convert UV transilluminator to Blue light transilluminator using a UVP VISIBLUE CONVRTR PLATE, 21X26 cm. This item can be purchased from VWR (Cat# 15000-088, $318.00).
Visi-Blue Converter Plate. 21Wx26Lcm. Designed to convert UV light to 480nm blue light for use with GR Safe, SYBR Green, SYPRO Orange and EGFP stains. Scratch resistant blue Plexiglas surface is safety fused into metal frame for durability. For UVP transilluminators.

6, Is is safer than SYBR Safe?

Yes or equivalent, but better and cheaper.

7, Is it more sensitive than SYBR Safe?

Yes or equivalent, but better and cheaper.

Other Related Items

1, New unused Qiagen QIAprep Spin Plasmid Miniprep Kit, 250 spin columns For 250 purifications of up to 20 ug molecular biology grade plasmid DNA.

2, New Qiagen EndoFree Plasmid Maxi Kit (10), Cat # 12362 containing 10 QIAGEN-tip 500, Reagents, 10 QIAfilter Maxi Cartridges, Endotoxin-free Buffers.

3, New QIAGEN Plasmid Maxi Kit (10), Cat # 12162, containing 10 QIAGEN-tip 500, Reagents, Buffers.

4, New Qiagen RNeasy Mini Kit (50), Cat # 74104, containing 50 RNeasy Mini Spin Columns, Collection Tubes (1.5 ml and 2 ml), RNase-free Reagents and Buffers.

5, New unopened Invitrogen Lipofectamine 2000, Cat # 11668-019, 1.5 ml,

Technorati : DNA, Ethidium Bromide, Nucleic Acid Stain, RNA, SYBR Gold, SYBR Green, SYBR Safe

Technorati : DNA, Ethidium Bromide, Nucleic Acid Stain, RNA, SYBR Gold, SYBR Green, SYBR Safe

A. Checking Peptide Thiol Groups

Do this before thiol coupling, either to KLH or to resin. Peptide thiol groups have a tendency to get lost after synthesis.

1. Make up 5 mM Ellman’s reagent (dithio-bis-2-nitrobenzoic acid) in 0.1M NaPi pH 7.2.
2. Weigh out about 1 mg of peptide into a tared tube.
3. Add 0.5 ml reagent. It should go bright yellow.
4. Dilute the mixture 1/50 in buffer. Read A412 against reagent at the same concentration.
5. Calculate the apparent molecular weight of the peptide based on thiol groups, using a molar extinction coefficient of 14,000. Compare this to the expected molecular weight of the peptide. They should agree within a factor of three, with the apparent molecular weight usually higher. If the thiol concentration is anomalously low, i. e., the apparent molecular weight is very high, there may be something wrong with the peptide- anyway it probably will not couple well. You may need to regenerate the thiol groups by reducing the peptide with excess DTT and running a P2 column.

B. Coupling of Peptide to KLH

This recipe is for two bunnies for about five injections per bunny

1. Weigh out 100mg of keyhole limpet hemocyanin (KLH). Dissolve in 2ml water. It generally takes about 4 hours to dissolve- you will need to sonicate and vortex. Be patient and put on a rotator at 4 deg C. Dialyze against 2l of 0.1M NaPhoshate pH 7.8 overnight. This is to remove any contaminating thiols or amino compounds.
2. Spin 10 minutes at full speed in microfuge to remove aggregates (don’t be surprised to see a substantial pellet).
3. Split the KLH into 2 aliqouts for -SH and -NH2 coupling.
4. For -NH2 coupling, add 5mg peptide to one aliquot, followed by glutaraldehyde to 0.1% final. Add the peptide as a solid if it is soluble, otherwise from a 100 mg/ml stock in DMSO. Precipitation does not matter and often happens. After adding the glut, check the pH with pH paper, and adjust to 7.8 if necessary using NaOH. Incubate 8-12 hrs at 4 degrees, rotating gently.
5. Add a tiny pinch of NaBH4 to kill remaining glut. Make sure the sample is in a large tube since it tends to fizz up. Incubate 8-12 hrs at 4 degrees. This is the glut conjugate.
6. For the -SH coupling, warm the other aliquot of KLH to room temp. Add 1/9 th volume of Iodoacetic acid N-hydroxysuccinimide ester at 100mg/ml in DMSO. Make the DMSO stock fresh, and protect the iodoacetamide reagent from light. We make our own IAA-NHS ester, but it can be purchased from Sigma.
7. After 10 minutes at room temp the KLH will start to get a little cloudy. Load it onto a P-10 column equilibrated with 0.1M NaPhosphate pH 7.8. Make sure the column is at least 10 times the volume of the sample. Pool the KLH containing fractions by color (it will be sort of greyish green). Add 5mg of peptide to them, as in step 5 above. Incubate at least 8 hrs at 4 degrees, rotating gently.
8. Pool the coupled peptide from the two proceedures. Dilute to 5ml with 0.15M NaCl. If there is a precipitate, sonicate vigorously to break it up. Split the immunogen into 1 ml aliquots (each aliquot will immunize two rabbits) and freeze it.