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KENDRICK LABS CATALOGCatalog PDFCall 800-462-3417 or email 2d@kendricklabs.com for a price quote or to receive printed information by mail.2D ELECTROPHORESISMK-1 Mailing Kit
This
kit contains ten 1 ml tubes of Urea Sample Buffer, ten 1 ml tubes of SDS Boiling
Buffer; ten 1 ml tubes of SDS Boiling Buffer minus BME; ten 1 ml tubes of
Osmotic Lysis Buffer; ten 100 μl tubes of 10X Nuclease Stock; three 100 μl
tubes of Protease Inhibitor Stock Solution and two 100 μl tubes each of Calbiochem phosphatase inhibitor cocktail sets I and II (cat No 524624 & 524625). The compositions of these solutions
is given in the Kendrick Labs booklet "Suggestions for Sample
Preparation". In addition a Styrofoam mailer, inner carton for radioactive
samples and mailing instructions are included.
2D-ES-1: 2-D ELECTROPHORESIS BASIC SERVICE
Our standard gel
size is perfect
for immunoprecipitations, subcellular fractionations, most bacterial
preparations and many proteomics
applications. One Coomassie blue stained, dried 2-D gel of dried
dimensions 15 cm wide x 13 cm long, is returned for each sample along with a pH
gradient plot and a method description sheet suitable for reports or
publications. The example below is from an Escherichia
coli sample prepared in our Urea sample buffer (left) and SDS Boiling
Buffer (right) run identically on a pH 4-8 IEF gradient and 10% acrylamide slab
gel. The great advantage of SDS buffer is ease of sample preparation. Molecular weight standards (220, 94, 60, 43,
29, and 14 kDa) appear as bands on the right side of the gel labeled with
press-on numbers. One isoelectric point marker, added to each sample as an
internal standard, is marked with an arrow. Our standard IEF tube gel contains
2% pH 3.5-10 mix4L or 2% 4-8 mixed ampholines; transparency drying (STP-8) is standard. The duplicate
gel rate is used for duplicate gels with different stains and also for blots,
but not for different gel conditions. More photos of example gels.
See 2D-ES-5 for our large format option. 2D-ES-1: 1 sample, $175 2D-ES-1A: 2-3 samples, $165 each 2D-ES-1B: 4-8 samples, $155 each 2D-ES-1C: 9-20 samples, $146 each 2D-ES-1D: 21-200 samples, $140 each 2D-duplicate: $85 each More than 200 samples - Inquire
CUSTOM PH GRADIENTS are available using ampholines of pH 3.5-10, 2.5-5, 4-6 and 7-9. For basic proteins with pI's greater than 9, non-equilibrium pH gradient electrophoresis is used (NEPHGE, O'Farrell, P. et. al. Cell 12: 1133, 1977). Buffers and electrodes are reversed and the pH gradient is created using pH 8-10.5 ampholines. Indicate which custom pH gradient you need on the back of the sample ID form. (No extra charge) 2D-ES-2: 2D ELECTROPHORESIS WITH PEPTIDE SLAB
GELS. The 16.5% peptide slab gels of Schagger and von Jagow (Anal. Biochem.
166: 368, 1987) are useful to resolve low MW proteins (3 - 14 kDa). In
general it is better to load low MW proteins heavily (10 μg/spot). Note
that ampholines may interfere in the MW range 2-6 kDa. 2D-ES-3: NATIVE 2D ELECTROPHORESIS For
native 2D the urea and IGEPAL (NP-40) are omitted from the IEF tube gel; SDS and BME
are omitted from the 2nd dimension slab gel. No denaturing agents are used
in the sample buffer. Many proteins are insoluble under these conditions and
don’t resolve. However, occasionally it’s very useful for soluble proteins in
serum and CSF. 2D-ES-4: 2-D ELECTROPHORESIS WITHOUT
SULFHYDRAL REAGENT Same as 2D-ES-1 except
2-mercaptoethanol and dithiothreitol are omitted from sample buffer,
internal standards, and all 2-D solutions.
2D-ES-5 LARGE FORMAT 2D
ELECTROPHORESIS The larger size gives better resolution for
proteomics applications; heavier loads may be applied to obtain more
material for mass spectrometry. One Coomassie blue-stained, dried 2D gel
of dimensions 20 cm wide by 20 cm long is returned for each sample along
with a pH gradient plot and a method description sheet. Our
standard IEF tube gel contains 2% pH 3.5-10 mix4L or 4-8 mix ampholines. Molecular
weight standards (220, 94, 60, 43, 29, and 14 kDa) appear as bands on
the right side of the gel labeled with press-on numbers. One IEF marker,
added to each sample as an internal standard, is marked with an arrow.
Transparency drying (STP-8) is standard. See above for Custom
pH gradients. Typical protein loads are 100 (silver staining) or 600
μg
(Coomassie blue) in 25-150 μl as shown in images below. 2D-ES-5: 1 sample $245 2D-ES-5A: 2-3 samples $235 each 2D-ES-5B: 4-12 samples $225 each 2D-ES-5C: 13-200 samples $215 each LF-Dup (Large Format 2D duplicate gels): $100 each More than 200 samples - Inquire.
2D-ES-6: Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a charge shift method developed by Schagger and von Jagow (Anal. Biochem. 199: 223-231, 1991) for isolation and characterization of large multi-protein complexes (MPCs) in their native state. Binding of Coomassie blue G-250 in the absence of denaturing detergents allows for first dimension separation according to the size and shape of the complex on 4-13% gradient gels.
Coupling first
dimension BN-PAGE with a second dimension of SDS-PAGE allows for the
dissociation of individual subunits based on molecular weight. Further
analysis by Western blotting or mass spectrometry can be performed to
identify specific subunits within MPCs. We currently offer both BN-PAGE
as the first dimension only, or BN-PAGE followed by SDS-PAGE as a second
dimension. BN/SDS PAGE has proven a valuable tool for studying the
functional proteomics of mitochondrial protein complexes (Methods 26:
327-334, 2002), membrane protein complexes (Anal. Biochem: 217: 220-230,
1994), and whole cell lysates (Molecular and Cellular Proteomics: 3.2:
176-182, 2004). Please call to discuss before sending samples for BN-PAGE.
2D-ES-6: 1 sample $175 2D-ES-6A: 2-3 samples $165 each 2D-ES-6B: 4-8 samples $150 each 2D-ES-6C: 9-20 samples $140 each 2D-ES-6D: 21-200 samples $130 each
BN-PAGE (1 D gel only): $110 first sample BN-PAGE-A: $50 each additional
2D-ES-7: CARBAMYLATED CARBONIC ANHYDRASE pI MARKERS This protein was purchased from Sigma-Aldrich, progressively carbamylated on lysine residues by heating in urea buffer for various times, and calibrated for pI. Each spot in the beads-on-a-string pattern differs from the next by a single charge; measured pI values are shown in the table. A pH gradient standard curve may be determined from the train of charge isomers added an an internal standard for calculation of pIs for unknown proteins. Note that these pIs are not absolute values but are for conditions of 9 M urea and 22o C. The arrow marks an internal standard protein, tropomyosin, MW 33, 000, pI 5.3. The right-most spot is 1, pI 7.3. Price: Add $16 per gel.
2DE/Top Mass Spectrometry Staining Blotting Radioactivity Sample Preparation Misc
2D-ES-9: MANUAL COMPARISONS of 2D PATTERNS FOR
DIFFERENCES. We find that HBC (Human Brain Comparison)
is a reliable method for finding changes in 2D patterns. In this method two
experienced analysts independently compare stained gel pairs on a light box for
differences. Results are presented as color coded outlines of spots on
transparent overlay covering the gels. Free duplicate gels are run to
confirm differences for samples scheduled for manual comparisons. 2D-ES-9: $165 per Standard Format pair compared 2D-ES-9LF: $195 per Large Format pair compared
2D-ES-10: COMPUTERIZED COMPARISONS OF 2D GEL PATTERNS FOR DIFFERENCES. This analysis includes scanning with a calibrated densitometer and computerized analysis with SameSpots software v4.0 (2010) from Non-Linear Dynamics. Requires prior 2D electrophoresis but free duplicate gels are run and analyzed to confirm differences for samples scheduled for computerized comparisons. Generally 400-800 spots are quantified per Standard Format gel; 800-1200 spots are quantified per Large Format gel. Spot density values are expressed as spot percentages (individual spot density as a percentage of total density in all spots analyzed) to normalize for differences in sample loading or staining.
Results are presented in a complete report including a
summary table showing spot number, pI, MW, ratios (fold difference) and p
values as a second measure of difference. The final report also includes
figures of images showing numbering, montage images for every differing
protein spot, methods and pH gradient plot. A CD containing data and image
files is included in the package. Note that our reports focus on the protein
differences between samples. Picking protein spots later for identification
by mass spectrometry is straightforward. Just send us a list of spot numbers
are we'll take it from there. Price: Inquire for project-specific quote. 2D-ES-10 (Standard Format): duplicate gels analyzed and averaged per sample. Per comparison, $450. 2D-ES-10LF (Large format): duplicate gels analyzed and averaged per sample. Per comparison, $500.
2D-ES-12 QUANTIFICATION OF PROTEINS RESOLVED BY 2-D ELECTROPHORESIS.
For clients knowing which proteins need to be quantified. Scanning (2D-ES-13)
is included. Data is reported in integrated spot density in density units,
or as a percentage of total image density.
Spots are not matched from gel to gel. 2D-ES-12: 1-12 spots $85 2D-ES-12A:13-50 spots $145 2D-ES-12B:1-200 spots $180 2D-ES-12C:1-400+ spots $285
2D-ES-13: LASER DENSITOMETRY. Stained gels and x-ray films are digitized with our laser densitometer from Molecular Dynamics. The densitometer is linear over the range 0-3.0 optical density units as verified by calibrated filters. Narrow O.D. ranges can be expanded to full scale (8 bit, 0-255) if required. Scanning resolution is 100 microns/pixel; the final black & white tif images are 3.3 Mbytes each for standard format and 7 Mbytes for large format. Price: $25/scan.
Price: $35/scan
Price: $25/scan 2D-ES-14: ELECTRONIC PHOTOS AND FILES. The 10 mB
black & white files obtained from laser densitometry are too big to
email and sometimes don't look right by eye. Our Afga Arcus II and HP
desktop scanners give
excellent color images in tif format that are useful for publication and
emailing (jpg format) The price includes tif and jpg image files on a CD
mailed with the dried gels, and jpg images emailed to provide previews of 2D
patterns. 2DE/Top Comparisons Staining Western blotting Radioactivity Sample Preparation Misc Protein Identification by Mass Spectrometry We have considerable experience in sending 2D gel proteins to university and commercial core facilities for identification by mass spectrometry fingerprinting (also called peptide mass matching) and for amino acid sequencing. Turnaround time for mass spectrometry is usually about two weeks. We highly recommend Columbia University Protein Core Facility Contact Dr. Mary Ann Gawinowicz, the facility director, for more information. tel: 212-305-3631, email: mag4@columbia.edu 2D-ES-19 Spot cutout for mass spectrometry Fingerprinting or sequencing We do this manually and are obsessively careful. The cutout spots are placed in Eppendorf tubes for express mail to the core facility of your choice; our default is Columbia University. The cost includes a letter to the core listing the spots, plus a copy of the letter for you along with an image or original gel showing spot locations. We make sure that each spot has a unique identifier. Price: $20 per spot
2D-ES-15 MASS SPECTROMETRY FINGERPRINTING (peptide mass matching):
This technique is used to identify proteins from Coomassie blue or special
silver-stained gels. The protein must be known and in the databases. The
cutout polypeptide spot is digested with an enzyme of known specificity,
usually trypsin, and the resulting peptide mixture, without separation,
is analyzed by MALDI (matrix-assisted laser desorption ionization) MS.
The peptide masses obtained by MS are entered into a search program
which scans the database, NCBI or Genpept, to find a match. This method
will not produce sequence data from the protein, only a unique set of
peptide masses enabling identification. The mass spectrum will usually
give 25-60% sequence coverage (number of amino acids in identified
peptides/number of amino acids in whole protein). Search
programs used at the Columbia Protein Core Facility are ProFound at
http://prowl.rockefeller.edu/prowl-cgi/profound.exe MS-Fit at
http://prospector.ucsf.edu , and Mascot at
www.matrixscience.com
Protein spots are cut from Coomassie blue
or special silver-stained gels and digested with trypsin. The digestion
mixture is separated on an LC Packings nano-lc on which the detector outlet
is connected directly to the nanospray source of a Micromass Q-Tof mass
spectrometer. Peptides are eluted at a flow rate of 200 nl/min and are
scanned as they enter the source. When a peptide is detected, the Q-Tof is
programmed to switch to MS/MS mode, which means that the eluting peptide is
fragmented by means of an applied collision energy and the resulting ions
scanned for several seconds. When the programmed time for MS/MS is done, the
Q-Tof switches back to MS mode and resumes scanning the eluting peptides.
Because peptides fragment at the peptide bond in a predictable way, the
fragmentation pattern can be used to deduce sequence information. The ions
resulting from fragmentation along with the mass of the intact peptide can
be used to search a database and identify the protein. Proteins which show a
good MALDI pattern but cannot be identified by Mass Spectrometry
Fingerprinting (2D-ES-15) are candidates for identification by LC/MS/MS.
Generally, LC/MS/MS is not more sensitive than MALDI, but the data is
acquired differently and can be more informative.
Reference for protein identification
by LC-MS/MS
2D-ES-20 PHOSPHORYLATION SITE IDENTIFICATION BY MASS SPECTROMETRY.
We
outsource this state-of-the-art service to the experts at the Laboratory
for Proteomic Mass Spectrometry at the UMass Med School. See:
www.umassmed.edu/proteomic/Leszyk/index.aspx for more information.
Contact Dr. John Leszyk, Tel: 508-856-7533 Email:
John.Leszyk@umassmed.edu
Note that
the success of phosphoprotein analysis depends on the type of protein
and the extent of phosphorylation. In some cases additional work may be
required. Note that the success of phosphoprotein analysis depends on
the type of protein and the extent of phosphorylation. See our
Feb 09
Newsletter for a brief literature review and advice on amount
of phosphorylated protein required. 2D-ES-17
N-TERMINAL AMINO ACID SEQUENCING BY EDMAN DEGRADATION. Typically done after 2D
electrophoresis and PVDF transblotting to determine if a protein is
clipped. The protein must be loaded
heavily enough to see with Coomassie blue on the blot; the N-terminus
cannot be blocked. Note that bacterial proteins are seldom blocked
at the N-terminal while mammalian proteins are often blocked, up to 60-70%
of the time. This is older technology but useful in some cases, for
example to see if a protein has been clipped by a protease.
2D
Gels/Top
Comparisons
Mass Spectrometry
Blotting
Radioactivity
Sample Preparation
Misc STAINING/DRYING PROCEDURESSTP-1: COOMASSIE BLUE STAINING. We use Coomassie brilliant blue R250 as our standard staining technique (O'Farrell, P. J. Biol. Chem. 250: 4007, 1975). This method, performed according to SOP L-1510, involves an alcohol/acetic acid fix, an acetic acid rehydration and an acetic acid destain. Generally, 1 µg of purified protein gives a nice spot on a 2D gel (see our internal standard) and 0.1 ug is visible. Although not as sensitive as silver staining, this method gives quantifiable results. (Burgess-Cassler et. al. Clin. Chem. 35: 2297, 1989.) The linear range for plots of stain density versus ng protein is broad and the method is reproducible. This stain is compatible with mass spectroscopy. Price: No charge (This is our standard stain).
STP-2: SILVER STAINING. Our silver stain is based on the ammoniacal silver/formaldehyde
method of Oakley et. al. (Anal. Biochem.105: 361, 1980.) This method
involves preliminary glutaraldehyde treatment of the slab gel to fix proteins by
cross linking. The pre-treatment also adds glutaraldehyde side chains to the
proteins, increasing sensitivity since these groups are sites for silver
deposition (Dion A, and Pomenti A, Anal. Biochem. 129: 390, 1983). We
find this method to be about 10 times more sensitive than Coomassie blue
staining, depending on the protein, even though less protein is loaded.
Generally, 50 ng of purified protein gives a highly visible spot on a 2D gel
(see our internal standard). However, some proteins that are detectable with
Coomassie don't stain at all with silver. Although very sensitive, this
method gives semi-quantitative rather than quantitative results. The linear range for plots
of stain density versus ng protein varies widely from protein to protein and
also because day to day variability in stain and background intensity is
observed. Silver staining is not compatible with mass spectroscopy
fingerprinting. STP-2 (Standard Format): $32 per gel STP-2LF (Large Format): $42 per gel
STP-3: SPECIAL SILVER STAINING FOR MASS
SPECTROSCOPY ANALYSIS. This special
silver stain (O’Connell and Stults, Electrophoresis 18: 349-359, 1997) omits the
glutaraldehyde step and is compatible with mass spectroscopy fingerprinting. It often negatively stains
proteins and so is not appropriate for computerized analysis. However, the pattern can be matched to our regular stain without
difficulty. Note however, that silver deposition interferes with the enzymatic
digestion required for mass spectrometry for most mammalian proteins. If possible use the less sensitive but
more reliable Coomassie stain. STP-3 (Standard Format): $32/gel STP-3LF (Large Format): $42/gel
STP-4: SYPRO RUBY FLUORESCENT STAIN.
This Molecular Probes stain fluoresces linearly with protein over a range of
100-1000 ng and is compatible with mass spectrometry. See 2D-ES-13A
Fluorescent densitometry for imaging. STP-4 (Standard Format): $65 per gel STP-4LF (Large Format): $90 each
STP-5:
Cy dye staining for DIGE analysis
STP-6: EMERALD GLYCOPROTEIN FLUORESCENT STAIN
This Molecular Probes stain is specific for glycoproteins and also compatible with mass spectrometry
but isn't very sensitive. See 2D-ES-13A Fluorescent densitometry
for imaging. STP-6 (Standard Format): $105 per gel STP-6LF (Large Format): $210 per gel
STP-7: PRO-Q DIAMOND STAIN FOR PHOSPHOPROTEINS This Invitrogen stain uses a novel fluorophore that recognizes phosphate groups on proteins directly on gels - without antibodies or radioactivity. Although many groups have had success with this stain we cannot guarantee sensitivity or specificity. See 2D-ES-13A Fluorescent densitometry for imaging. Price: STP-7SF (Standard Format): $60 per gel STP7-LF (Large Format): $75 per gel
STP-8: TRANSPARENCY DRYING. All stained gels are air dried between cellophane sheets unless otherwise requested. Although this treatment adds a day to turnaround, the advantages of easier storage, little or no curling, good color retention, overlay capability, scanning capability, and overhead projection capability offset the time delay. Since cellophane quenches 35S and 3H, gels with these isotopes scheduled for autoradiography or fluorography are paper dried (STP-9). Price: No extra charge.
STP-9: PAPER DRYING This is an optional drying method for stained gels. All Enhance-treated slab gels scheduled for fluorography as well as unstained gels scheduled for autoradiography are dried onto thick filter paper unless otherwise requested. Price: No extra charge. STP-10: WET GELS In some cases the return of wet gels is requested, for example, for a core facility's automatic spot picker. This is no problem. The wet gels are placed between sheets of thick wet filter paper and the sandwich placed in a ziploc bag. The bag is supported by pressboard sheets and cushioned by bubble wrap prior to express mailing. Gel identification information is written on the ziploc bag. Price: No extra charge. 2D Gels/Top Comparisons Mass Spectrometry Staining Blotting Sample Preparation Misc
RADIOACTIVITY PROCEDURESKendrick labs has a Radionuclide License (NRC) that can be faxed or emailed to your RSO to ease the shipment of radiolabeled samples. See page 3 for radioisotope mailing instructions. RP-1: AUTORADIOGRAPHY.
Includes 2 film exposures at room temperature using 8" x 10" Kodak BioMax MR
film. Exposure times are usually 2 and 4 days. RP-2: ENHANCE TREATMENT.
For
fluorography of low energy emitters such as 3H,
14C, and 35S. (New England
Nuclear reagent and protocol). The gel is soaked in a solution (Enhance)
containing a fluorescent intermediate which, at low temperatures, converts
beta-particle energy into photons. This treatment reduces exposure times for
14C and 35S to about 1/8 that of untreated gels. Note that disposal costs
for used Enhance add to the cost. RP-2 (Standard Format): add $45 per gel RP-2LF (Large Format): add $65 per gel RP-3: FLUOROGRAPHY.
Requires
prior Enhance treatment (RP-2). Includes 2 film exposures at -70ºC using gel
8" x 10" Kodak BioMax MR film. Exposure times are usually 2 and 4 days. RP-4: 14C-MOLECULAR WEIGHT MARKERS.
These markers from Amersham are added during slab gel electrophoresis so that
bands appear on X-ray film after exposure of the gels. These markers have molecular weights of 200, 97.4, 69, 46, 30 and 14.3
kDa.
RP-6 RADIONUCLIDE DISPOSAL FEE
to help cover the high cost of our Radioactive Materials License and disposal fees. 2D Gels/Top Comparisons Mass Spectrometry Staining Radioactivity Sample Preparation Misc
BLOTTING & WESTERN BLOTTING P-Tyr WB package P-Ser/P-Thr WB packageBP-1: TRANSBLOTTING ONTO PVDF.
PVDF (Immobilon
from Millipore Corp.) binds proteins more tightly than nitrocellulose
and gives better recoveries. Transblotting onto PVDF is carried out
overnight according to standard procedures using a tris/glycine/methanol
buffer system without SDS. The dried stained (see BP-2) or unstained blot is
returned to the client. Staining facilitates matching to a duplicate Coomassie gel for mass spectrometry. Original gels are stained to make sure
they’re blank and discarded, unless otherwise requested. We will change the
blotting procedure to your specifications if necessary. BP-1 (Standard Format): $70 per blot BP-1LF (Large Format): $80 per blot BP-2: PVDF COOMASSIE STAINING.
The 2D gel pattern obtained from Coomassie blue staining of a
PVDF membrane is faded but the scanned image (2D-ES-14, Electronic photos,
$15) can be contrasted with Adobe photoshop. The image is super-imposable with the ECL
film from Western blotting and helps to match a few spots on the film to a
busy 2D gel pattern on a duplicate Coomassie gel run for mass spectrometry.
Since Western blotting can be 100 times more sensitive than Coomassie, exact matching is
very important. Coomassie staining
of the blot does not interfere with subsequent Western blotting. BP-3: TRANSBLOTTING ONTO NITROCELLULOSE.
Transblotting is carried out overnight according to standard
gel procedures using a tris/glycine/methanol buffer system containing 0.1%
SDS, with nitrocellulose of 0.45 micron pore size. Dried unstained blots are
returned to the client and original gels are discarded unless additional
treatments are requested. We will change the blotting procedure to your
specifications if necessary. BP-3 (Standard Format): $70 per blot BP-3LF (Large Format): $80 per blot BP-4: COLORED MOLECULAR WEIGHT MARKERS FOR BLOTS.
These markers from Diversified Biotech are added during slab gel
electrophoresis; so that colored bands are visible on both unstained gels
and subsequent transblots at molecular weights of 95.5, 55, 43,
36, 29, 18.4, and 12.4 kDa. BP-5: UBIQUITIN WESTERN BLOTTING PACKAGE INCLUDING ANTIBODY. We have optimized a method for identifying ubiquitin-containing proteins by 2D Western blotting using the Bethyl Labs anti-ubiquitin antibody (A300-317A). Our results suggest that the method is specific, reproducible, and sensitive. Note that this protein is very highly conserved so the antibody should react with all species. Price: BP-5SF (Standard Format Package): $325/sample includes standard format 2D gel electrophoresis, transblotting to PVDF, blot Coomassie staining, subsequent immunostaining with anti-ubiquitin antibody, and electronic photos of stained blot and ECL film. BP-5SF-Dup (Replicate SF Package): $175 each BP-5LF (Large Format Package): $400 BP-5LF-Dup (Replicate LF Package): $220 each
BP-6: WESTERN (IMMUNO-) BLOTTING, CLIENT SUPPLIES ANTIBODY. This method for immuno-detection of specific bands or spots is carried out on PVDF transblots from 1-D or 2-D gels (see: "Handbook of Immunoblotting of Proteins Vol 1 and 11", Eds. Bjerrum, O. and Heegaar, N., 1988, CRC Press, Inc; and "Gel Electrophoresis: Proteins" by Dunn, M.J., 1993, Bios Scientific Publishers, Ltd). Requires prior electrophoresis (2D-ES-1 or 1D-ES-1) but PVDF transblotting (BP-1) is included in the fee. Generally the client supplies the antibody. The sensitive ECL method of detection is used. (Amersham reagents and protocol are used.) A control membrane run with secondary antibody is included as a control if necessary. BP-6SF Standard Format: Add $275/sample BP-6SF-Dup: Add $100 BP-6LF Large Format: Add $350/sample BP-6LF-Dup: Add $140
BP-7:
PHOSPHOTYROSINE WESTERN BLOTTING PACKAGE INCLUDING PY20 ANTIBODY. We have
optimized a method for
identifying phosphotyrosine-containing proteins by 2D Western blotting
using the PY20 anti-PTyr antibody. Our results suggest that the method is
specific, reproducible, and sensitive. However, in some cases the phosphate
bond is labile so duplicate Western blots are encouraged to verify
reproducibility. Mass spectrometry to identify proteins found by Western blotting is extra
and requires a duplicate Coomassie blue stained gel. Ask for a
quote by calling
800-462-3417 or emailing
2d@kendricklabs.com BP-7SF (Standard Format): $325/sample includes standard format 2D gel electrophoresis, transblotting to PVDF, blot Coomassie staining, subsequent immunostaining with PY20 antibody, and electronic photos of stained blot and ECL film. BP-7SF-Dup (Replicate SF Package): replicate W blot package $175 each BP-7LF (Large Format Package): $400 BP-7LF-Dup (Replicate LF Package): $220 each
BP-8: COMBINED P-SERINE/P-THREONINE WESTERN BLOTTING PACKAGE USING ECL ADVANCE AND QIAGEN’S Q5 AND Q7 ABS. We have optimized a method for 2D Western blotting against phosphoserine and phosphothreonine residues on proteins using the Q5 and Q7 antibodies from Qiagen and ECL Advance from GE Healthcare. Both antibodies are advertised as detecting phosphorylated residues irrespective of surrounding amino acids. Note that non-specific binding shouldn't matter if control and test samples are compared for differences - it'd be the same in both. The ultra sensitive ECL Advance enables us to dilute the antibodies to 1:4000 and combine them for this package. The high sensitivity adds variability so all the BP-8 packages include duplicate Western blots to verify results. BP-8SF (Standard Format): $575 includes duplicate SF 2D gel electrophoresis, duplicate transblotting to PVDF, blot Coomassie staining, subsequent immunostaining with combined Q5/Q7 antibodies, and electronic photos of stained blots and ECL films. BP-8SF-Dup (Replicate SF Package): $200 each including 2D gel BP-8LF (Large Format Package): $750 for package including duplicate blots BP-8LF-Dup (Replicate LF Package): $275 each
BP-9: PHOSPHOSERINE OR PHOSPHOTHREONINE WESTERN BLOTTING.
Same package as above except single antibodies are used instead of a
mixture. BP-9SF (Standard Format): $520 includes duplicate blots BP-9SF-dup (SF Replicates): $180 each BP-9LF (Large Format): $675 includes duplicate blots BP-9LF-dup (LF Replicates): $250 each
BP-10: Acetyl-lysine Western Blotting. Acetylation is an important post translational modiciation occuring not only on histones but many other proteins. 2D electrophoresis and subsequent western bloting is a powerful tool for identifying acetylated proteins. Using Cell Signaling Technologies polyclonal acetylated lysine antibodies western blotting is carried out in a similar manner to the Ubiquitin western blotting package (BP-5). BP-10SF (Standard Format) package: $325/sample includes standard format 2D gel electrophoresis, transblotting to PVDF, blot Coomassie staining, subsequent immunostaining with anti-acetylated lysine antibody, and electronic photos of stained blot and ECL film. BP-10SF-Dup (SF Replicates): $175 each BP-10LF (Large Format) package: $400 same package as BP10-SF except LF 2D gels are run. BP-10LF (LF Duplicates): $220 each
Right: Acetylated proteins in rat liver. The anti-acetyl-lysine antibody from Cell Signaling was used for 2D gel WB at a 1:1000 dilution with overnight incubation. The ECL film image from Western blotting is shown with permission of Blythe Shepard and Dr. Pamela Tuma, Catholic University of America, Washington, DC. See also: Shepard, Blythe D., Dean J. Tuma, and Pamela L. Tuma. "Chronic Ethanol Consumption Induces Global Hepatic Protein Hyperacetylation." Alcohol Clin Exp Res 34.2 (2010): 1-12.
2D
Gels/Top
Comparisons
Mass Spectrometry
Staining
Blotting
Radioactivity
Misc
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