rev: May 14, 2008
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PROTOGOLD :Stabilised Colloidal Gold Sol for the sensitive stainging of protein blots.
1. Description
Protogold Colloidal Gold Sol has been specially developed to give intense staining of proteins blotted onto membranes by dot blots or from electrophoretic gels. The gold particles are negatively charged and bind selectively to the blotted proteins with very low background absorption on the membrane. The specific staining of proteins occurs by hydrophobic and ionic interactions. The protein stain dark red through the accumulation of gold particles and the method produces a sensitivity greatly in excess of Coomassie Blue or silver staining in gels with no fading. When used in conjunction with the silver Enhancing Kit (Cat RDI-SEKB250, new cat#: 55R-SEKB250) a further enhancement of the signal by 10-100x is also achieved. The Protogold Sol is supplied in 500ml volume, sufficient to stain over 20 blots with high intensity. Each kit is supplied with 5ml of detergent to assist in the washing of blotted membranes before staining. The solutions should be kept at 4C and have a shelf life of 12 months.
2. Method of use
a). Handling membranes
Protogold is intended for use with negatively charged membranes such as
nitrocellulose or polyvinylidene difluoride (eg PVDF Immobilon-P from Millipore
Corp). Membranes should be handled with gloves and forceps to avoid smears.
Overloading the membranes with protein should be
avoided due to the high sensitivity of the staining procedure. Up to 1ug per
band or dot blot is recommended since higher concentrations may not be retained
by the membrane during the staining procedure. In this case spots will appear
smeared and will release protein into the sol where the colloidal gold will
aggregate and turn blue. It is recommended that membranes blotted with proteins
are washed in a solution of 0.3% detergent (supplied concentrated) and then in
distilled water in order both to block the unoccupied spaces on the membrane
and to remove damaging buffer salts from the membrane. Normal Western Blotting
procedures are recommended for transfer to the membrane either by
electrotransfer or by capillary blotting. After blotting the membrane should be
washed in phosphate buffered saline (PBS) at pH7.4 for 2 X 5 minutes before the
staining procedure.
b). Staining procedure
1) Incubate the blotted membrane with PBS containing 0.3% of the detergent to block unoccupied sites. Best results are obtained if this is done at 37 DEG C. The membrane is then washed again 3 X 5 minutes in PBS with 0.3% detergent at 20 EDG C. It is found that these steps will produce a significantly reduced background stain when looking for low levels of blotted proteins.
2) Wash the membrane in distilled or deionised water 3 X 1 minute to remove
buffer salts.
3) Incubate the membrane with the PROTOGOLD solution in a glass or plastic tray
with 2mm of solution covering the surface. The tray should be continuously
agitated. Alternatively membranes may be placed in sealed plastic bags and
agitated on a rocking machine for the period of incubation. It is important
that trays and utensils are clean and do not introduce either salts or proteins
into the solution. The staining of the protein takes place continuously and
shows as pink to scarlet stain on the membrane, increasing with time. Optimum
staining occurs between 2-4 hours, but the incubation can be continued
overnight or longer without overstaining.
4) After staining the membrane is washed in distilled or deionised water and dried. It may be kept indefinitely without fading.
C) Silver Enhancing
If required the membrane stain may be further enhanced by 100% or more by use
of the Silver Enhancing Kit (CAT RDI-SEKB250, new cat#: 55R-SEKB250). In this
case the membrane should be washed thoroughly in deionized or distilled water 5
X 2 minutes with agitation to remove loose gold. Even previously dried membrane
which had been gold stained may be enhances, but better results are obtained if
the membrane is silver enhanced immediately following gold staining and washing
in water. Silver enhance the membrane with similar incubating and agitating
procedures to the gold stain incubations. Enhancement takes approximately 10-20
minutes and the gold stain turns to an intense black at the sites of blotted
protein. The enhancement is stopped simply by washing the membrane thoroughly
in running tap water for 5 minutes. No fixing is necessary. Dry the membrane
between filter papers. The final stain is permanent.
3. Trouble Shooting
a) Protogold Gold Sol turns blue or colourless during the incubation.
This is due to excess protein coming off the membrane precipitating gold in solution. The membrane should be washed in distilled or deionised water for one minute and incubated with fresh PROTOGOLD solution.If nexessary increase the volume of PROTOGOLD for the incubation. Ideally the protein loading of the membrane sshould not exceed 1 microgram per band or dot blot. Where molecular weight standards are used in an electrophoresis run the maximum concentration should be 0.2 micrograms per band.On dot blots this limit may be tested by serially blotting 1ul blots of increasing protein along a strip of the membrane and staining it. Where excess protein occurs the membrane cannot retain it and smearing occurs outside the blot as well as discolouration of the gold solution. This loose protein may then attach non specifically to the membrane and cause background staining.
b) High background on the membrane.
1) This may be caused by the membrane being inadequately blocked before incubation with the gold sol. Ensure that the membrane is thoroughly washed in detergent solution (2(b) above) before exposing to PROTOGOLD.
2) Excess salts introduced into the PROTOGOLD incubating solution. Same procedure as for (1) above.
3) Excess protein contamination onto the membrane or into solutions. Use lower
protein loading of the membrane and avoid introducing protein contamination
from pipettes, fingers, etc.
4) Impure water used in the washing of membrane. Check the quality.
5) Excessive silver enhancing time. Reduce the time. Temperature can affect the
rate of enhancing. Inadequate washing of membrane before
silver enhancing.
C) Low signal of gold stain
1) Poor attachment of protein to the membrane. Check the blotting procedure.
Transfer from gels may be inadequate. Attachment of proteins to membrane may
vary with quality of membrane. Some membranes become hydrophobic if exposed to
light too long and will not absorb proteins.
2) PROTOGOLD sol is spoiled through contamination with proteins or excess
salts. Ensure that the staining solution is
stored at 4`C with cap securely screwed on. Adopt clean handling/pipetting
procedures.
3) Genuine low levels of protein. Incubate the membrane for 1-2 days to reveal
faint bands or spots and silver enhance. If necessary further enhance with
fresh silver enhancing solutions every 10 minutes. Faster staining of proteins
with PROTOGOLD and silver enhancing of the gold stain is sometimes achieved at
higher temperatures (eg 25-30`C).
Please ask for further details abount our complete immunogold and silver enhancing reagents for electron microscopy, light microscopy and blotting applications.
5. Ordering Information
PROTOGOLD is supplied in a kit containing 500ml of stablized gold sol, together with 5ml of concentrated detergent. It is shipped at ambient temperatures and should be stored at 4`C on arrival.
PRODUCT CODE: RDI-PRO500, new cat#: 55R-PRO500 $346.50/kit
RDI Division of Fitzgerald Industries Intl
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com