KENDRICK LABS CATALOG
Call 800-462-3417 or email email@example.com for a price quote or to receive printed information by mail.
MK-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
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.
photos of example gels. See 2D-ES-5 for our large format option.
2D-ES-1: 1 sample, $185
2D-ES-1A: 2-3 samples, $175 each
2D-ES-1B: 4-8 samples, $165 each
2D-ES-1C: 9-20 samples, $155 each
2D-ES-1D: 21-200 samples, $147 each
2D-duplicate: $90 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: Semi-native 2D
Electrophoresis: For semi-native 2D, denaturing agents urea, SDS, NP-40
and sulfhydral reagents are omitted from the sample buffer and from the
second dimension slab gel. However, 9M urea is a structural component of the
IEF tube gel and must be retained during isoelectric focusing. We don’t
recommend this. Proteins tend to clump under semi-native conditions; some
are insoluble and must be centrifuged off. However, in some cases this 2D
variation is useful.
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 $260
2D-ES-5A: 2-3 samples $245 each
2D-ES-5B: 4-12 samples $235 each
2D-ES-5C: 13-200 samples $225 each
LF-Dup (Large Format 2D duplicate gels): $105 each
More than 200 samples - Inquire.
Large Format 2D Gel Examples,
silver versus Coomassie staining. A side by side comparison of 2D gels
loaded with same sample and stained with Coomassie blue (left, 600 µg sample
load) and with silver (right, 100 µg sample load). The silver stain is at
least 10x more sensitive than Coomassie blue stain even though less is
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 $22 per gel.
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
2D-ES-9: $175 per Standard Format pair compared
2D-ES-9LF: $205 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 (13x15 cm); 800-1200 spots are quantified per Large Format gel (20x20 cm). 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, $475.
2D-ES-10LF (Large format): duplicate gels analyzed and averaged per sample. Per comparison, $525.
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 $90
2D-ES-12A:13-50 spots $155
2D-ES-12B:1-200 spots $190
2D-ES-12C:201-400+ spots $300
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.
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.
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: firstname.lastname@example.org
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: $21 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
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
Reference for protein identification
2D-ES-17 N-TERMINAL AMINO ACID SEQUENCING BY EDMAN
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.
STP-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): $35 per gel
STP-2LF (Large Format): $45 per gel
STP-3: SPECIAL SILVER STAINING FOR MASS
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): $35/gel
STP-3LF (Large Format): $45/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): $70 per gel
STP-4LF (Large Format): $95 each
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.
STP-7SF (Standard Format): $110 per gel
STP7-LF (Large Format): $225 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.
Kendrick 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.
Includes 2 film exposures at room temperature using 8" x 10" Kodak BioMax MR
film. Exposure times are usually 2 and 4 days.
For fluorography of low energy emitters such as
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
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 $50 per gel
RP-2LF (Large Format): add $70 per gel
Requires prior Enhance treatment (RP-2).
Includes 2 film exposures at -70şC using gel 8" x 10" Kodak BioMax MR film.
Exposure times are usually 2 and 4 days.
RP-4: 14C-MOLECULAR WEIGHT
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.
RADIONUCLIDE DISPOSAL FEE
to help cover the high cost of our Radioactive Materials License and
BLOTTING & WESTERN BLOTTING P-Tyr WB package P-Ser/P-Thr WB package
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): $75 per blot
BP-1LF (Large Format): $85 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
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): $75 per blot
BP-3LF (Large Format): $85 per blot
BP-4: COLORED MOLECULAR WEIGHT MARKERS FOR
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.
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): $190 each
BP-5LF (Large Format Package):
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 $290/sample
BP-6SF-Dup: Add $150
BP-6LF Large Format: Add $370/sample
BP-6LF-Dup: Add $200
PHOSPHOTYROSINE WESTERN BLOTTING PACKAGE INCLUDING PY20
ANTIBODY. We have optimized a method for
identifying phosphotyrosine-containing proteins by 2D
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
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
BP-7SF (Standard Format): $350/sample includes standard format 2D gel electrophoresis (13x15 cm), 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 $220 each
BP-7LF (Large Format Package): $425 includes everything in BP-7SF except the gels are large format (20 x 20 cm). Note that standard format 2D gels are 13 x 15 cm.
BP-7LF-Dup (Replicate LF Package): $265 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): $700 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-8LF (Large Format Package): $850 for package including duplicate blots on large format 2D gels (20 x 20 cm).
BP-9: PHOSPHOSERINE OR PHOSPHOTHREONINE WESTERN BLOTTING.
Same package as above except single antibodies are used instead of a
BP-9SF (Standard Format): $600 includes duplicate blots
BP-9LF (Large Format): $740 includes duplicate blots
BP-10: Acetyl-lysine Western Blotting. Acetylation is an important post translational modification occurring not only on histones but many other proteins. 2D electrophoresis and subsequent western blotting 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: $450/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): $300 each
BP-10LF (Large Format) package: $550 same package as BP10-SF except LF 2D gels are run.
BP-10LF (LF Duplicates): $400 each
Acetylated proteins in rat liver samples are shown above. 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."
SP-1: PROTEIN DETERMINATION
followed by buffer addition.
The total amount of protein in
each sample is determined by the Pierce BCA total protein assay. If you
require protein determinations, do not dissolve samples in buffers
containing dithiothreitol or B-ME. We will add B-ME after protein
SP-2 Combining multiple sample tubes, for
example, from an immunoprecipitation.
This involves transferring each
tube's contents, adding a rinse, vortexing, and transferring the rinse.
The rinsing step is repeated as needed.
SP-3: ETHANOL PRECIPITATION OF
followed by buffer addition.
Ethanol Ppt Link
SP-4: NUCLEASE TREATMENT
includes homogenization with osmotic lysis buffer containing protease
inhibitors followed by incubation on ice with DNase and RNase to break down
interfering polynucleotides. The composition of the 10X Nuclease Stock
Solution is given in our Sample Preparation Guide. If you require nuclease
treatment send the samples in a buffer containing less than 0.3% SDS. Higher
SDS concentrations inactivate nucleases.
followed by lyophilization & buffer addition.
High salt concentration (> 150 mM) interferes with IEF; the less salt the
better. If your samples contain high salt or buffer (phosphates,
NaCl, etc.), this dialysis and concentration step is highly recommended.
by buffer addition.
SP-7: TCA PRECIPITATION
followed by buffer addition.
followed by buffer addition.
SP-9: CUSTOM SAMPLE PREP.
(Chunks of tissue, leaves, etc.) Charged by the hour.
SP-11: ALBUMIN/IgG REMOVAL
FROM SERUM, PLASMA & CSF OF HUMAN, RAT AND MOUSE
using the Calbiochem
ProteoExtract Albumin/IgG Removal kit (Cat. No. 122642) Depletion of albumin
and IgG from 20-60 ul using the disposable gravity-flow affinity columns
removes up to 75% of total serum proteins so that 3-4 times more enriched
sample can be loaded on 2D gels.
CURRENT GOOD MANUFACTURING PRACTICE
The numerous SOPs required for cGMP for 1D and 2D electrophoresis are in
place at Kendrick Labs along with an archival system for equipment
calibrations and reports.
However, custom validation for the sample type of interest
including SOPs are required for new projects to pass detailed FDA
inspections - which may occur years down the road.
Please let us know if you are going to submit an IND, or plan
to use our lab for QA of a product.
CON-1 CONSULTING FEES Price: $190/hr.
Fish Species determination by the method
of Pineiro, C., Barros-Velazquez, R. Perez-Martin, I. Martinez, T. Jacobsen,
H. Rehbein, R. Kundiger, R. Mendes, M. Etienne, M. Jerome, A. Craig, I.
Mackie and F. Jessen, “Development of a SDS-polyacrylamide gel
electrophoresis reference method for the analysis and identification of fish
species in raw and heat-processed samples: A collaborative study”.
Electrophoresis, 20: p1425-32 1999.
HCP-LF Host Cell Protein Antibody Analysis:
Recombinant therapeutic proteins are
drugs produced by bioengineered bacteria or cultured cells. Over 100
recombinant proteins have been approved by the FDA for human use and many
more are being tested. Typically an Elisa assay is used to quantify host
cell protein (HCP) contamination of the final product.HCP-1 and HCP-LF are specialized 2DE tests
for characterization of anti-HCP antibodies chosen for the Elisa. In each
test, the number of spots on 2D ECL films from an optimized Western blot are
compared to the number on a silver-stained pattern from the same sample.
Light and dark films are used to create a detailed report. Antibodies
detecting a high percentage of the silver-stained proteins are desirable.
HCP ab Test link
provides more information.
Example: E. coli HCP Antibody Analysis
Final Result: 993/1400 total spots are detected by the antibody = 71% coverage. Individual protein spots of interest may be identified by mass spectrometry.