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FAQ
for Monoclonal Antibody Production>>
FAQ for Recombinant
Protein Production>>
Monoclonal Antibody Production
1. How log will
it take to get the antibody?
2. What positive
clones will I get?
3. Do I get the
hybridoma cell lines?
4. What kind of
antigens do you use to make antibodies?
5. How much antigen
is required for antibody development?
6. How
do you test antibodies that you have developed?
7. How
do you measure the antibody titer?
8. How should I
store my antibodies?
9. What is the
difference between the titer and the working dilution
of an antibody?
10. How do I determine
antibody concentration using the absorbance at
280nm?
11. What can I
use to block non-specific binding in immunological
techniques?
12. What is meant
by monospecific reactivity?
13. What's the
difference between a linear epitope and a conformational
epitope?
14. Why does one
see a very weak or no signal in Western blot?
15. Too many bands
on a Western blot?
16. Why do I observe
diffuse or moderate to intense staining of several
tissue elements by IHC?
Recombinant Protein Production
1. How many custom
proteins have you produced so far? What is the
% of successful productions?
2. What is guaranteed
provided at the end of production? Do we have
to pay even if there was no measurable protein
produced or if the protein is still very dirty
after your affinity purification?
3. Is payment
due upon delivery, or before start of the project,
or subject to milestones (e.g. 30%upfront,30 %upon
proofen production,30%upon delivery of purified
protein)?
4. Are purification
conditions native or denatured (we prefere native)?
5. Do you offer
a discount for His and GST tagged versions of
the same protein (normally 2 x 2300 $)?
6. Does the final
protein product have biological activities?
Monoclonal Antibody Production
1. How log will it take to get the antibody? ! TOP
It usually take three and half months to get the
first batch of antibodies in supernatant format.
If the antibodies will be used in research, we can
shipp this supernantant to you at 10 to 20 ml of
aliquot. Then we will do large scale production
in ascitic fluid.
2. What positive clones will I get? ! TOP
It is impossible to test the antibody's application
in every case, but we guarantee at least 10 positive
clones against customer-provided antigen in ELISA
assay. If customer wants more assay should be done,
talk to us for the extra services.
3. Do I get the hybridoma cell lines? ! TOP
At the end of each project, we will ship the subcloned
cell lines to you and we only keep the cell lines
up to 6 months without extra charge. If long storage
time is requested by customers, extra charge will
apply.
4. What kind of antigens do you use to make
antibodies? ! TOP
Promab's immunization protocols can be adapted for
native proteins, recombinant proteins, and peptides.
If the synthetic peptide is smaller than 8 kDa,
we conjugate the peptide to KLH through the thiol
on an added cysteine residue. Peptide
antigens have a lower success rate in eliciting
a strong immune response than do the larger recombinant
proteins or protein fragments. Peptides,
however, have the advantage that they can be carefully
designed to avoid sequence regions that are similar
or identical to other members of the same protein
family, thus making them potentially more specific.
5. How much antigen is required for antibody
development? ! TOP
If the antigen is a peptide, 5 mg free peptide and
5 mg conjugated peptide will generally be sufficient.
Less may be needed if your peptide is highly purified.
If the antigen is a protein, 3-5 mgs at a concentration
of 0.5-1 mg/mL is generally sufficient. Be sure
to let us know which buffer you included and the
protein concentration. In polyclonal antibody case,
For affinity purification of an antibody, 5 mg of
soluble protein is required to prepare the immunoaffinity
column.
6. How do you test antibodies
that you have developed? ! TOP
ELISA and western blot, and/or IHC and/or ICC depending
on antibody’s application have extensively tested
all of Promab’s antibodies. We can test antibodies
in other assays, like immunoprecipitation, flow
cytometry, upon customer request.
7. How do you measure the
antibody titer? ! TOP
Antibody titer is determined by ELISA. In this method,
we coat 96-well plates with antigen and wash repeatedly
to remove unbound antigen. Serial dilutions of antibody
are added to the antigen-bound wells and washed
repeatedly to remove unbound antibodies. A labeled
secondary antibody is then added and the amount
of bound primary antibodies is determined by a colorimetric
assay.
8. How should I store my antibodies? ! TOP
s Storage of lyophilized antibodies:
All our products are shipped lyophilized (freeze-dried).
In this form they are stable without loss of quality
at ambient temperatures for several weeks to months.
They can be stored at 40C for several years. After
reconstitution by addition of water, storage conditions
depend on the type of antibody, as described below.
Ascites fluid: When ascites fluid
is reconstituted, we add small amounts of azide
or thimerosal to prevent microbial growth. Ascites
fluid should be stored frozen. Monoclonals usually
do not lose an appreciable amount of activity from
freeze-thawing, but aliquoting them is recommended
to avoid repetitive freeze-thaw cycles. Prolonged
storage at 4oC is not recommended! Unlike serum,
ascites fluid may contain proteases that will ultimately
degrade the antibodies. Addition of protease inhibitors
helps to slow degradation.
Purified IgG:Do not store dilute
(<0.1mg/ml) antibody solutions unless carrier
proteins such as BSA is added. IgG’s, like other
proteins, bind non-specifically to glass and plastic.
Any IgG solution below 0.1 mg/ml protein will adsorb
to the storage vessel and denature and thus loose
activity! Repetitive freeze-thawing of dilute purified
IgG is almost certain to lead to substantial losses.
Rabbit serum: Antibodies in serum
are more robust than in ascites fluid. With anti-microbials
added, they may be stored at 4? (serum does not
contain significant protease activity; in fact,
serum itself contains a powerful cocktail of protease
inhibitors). Frozen storage, however, is preferable
for long periods (months to years).
Polyclonal affinity-purified antibodies:
Purified antibodies are less robust than in sera,
since protease inhibitors and carrier proteins are
removed during purification. Hence, storage at 40C
for prolonged periods (i.e. several weeks), is not
recommended.
9. What is the difference between the titer
and the working dilution of an antibody?
The titer of an antibody refers to the lowest concentration
(highest dilution) at which the antibody is still
effective in a given system. The working dilution
is usually the concentration which gives a good
sensitivity with a high signal : noise ratio. Using
too high a concentration is wasteful and may give
rise to non-specific signals. On the other hand,
using too low a concentration will lead to a loss
in sensitivity
10. How do I determine antibody concentration
using the absorbance at 280nm? ! TOP
The amount of antibody can be determined by absorbance
at 280nm. 1 OD = approximately 0.75 mg/ml of purified
antibody.
11. What can I use to block non-specific
binding in immunological techniques? ! TOP
This will largely depend on the type of antibody
used. For most ELISA and Western applications, 2%
non-fat milk is an excellent blocking agent. Alternatively,
3% bovine serum albumin is sometimes as effective.
Gelatin or serum (other than the species of primary
antibody) may also be used to block non-specific
binding.
12. What is meant by monospecific reactivity? ! TOP
A monospecific antibody is one that reacts with
only a single antigenic determinant. Monoclonal
antibodies are the only truly monospecific reagents.
The term is also used, however, to describe polyclonal
antibodies that were raised against and affinity-purified
against a peptide antigen.
13. What's the difference
between a linear epitope and a conformational epitope?
A linear epitope consists of about 6 to 10 adjacent
amino acids on a protein molecule that is recognized
by an antibody. In contrast, conformational epitope
consists of amino acids that are not arranged sequentially.
Here the antibody recognizes only the 3-dimensional
structure of the antigen. When a protein molecule
folds into a three dimensional structure, the amino
acids forming the epitope are juxtaposed, enabling
the antibody to recognize the sequence.
Knowledge of the differences between linear or conformational
epitope is of significance in immunological applications.
In a denatured protein, only the linear epitope
may be recognized. Hence in protocols where a denatured
protein is used, such as in Western blotting, an
antibody that recognizes a linear epitope is preferred.
Sometimes an epitope is inside the protein in its
native conformation. The epitope is then inaccessible
to the antibody in a non-denaturing protocol, such
as a non-denaturing immunoprecipitation. A conformational
epitope, by definition, must be on the outside of
the folded protein. An antibody that recognizes
the conformational epitope is suitable for mild,
non-denaturing procedures, such as non-denaturing
immunoprecipitations or flow cytometry.
Optimally, an antibody that recognizes a linear
epitope on the surface of a normally folded protein
will work well in both non-denaturing and denaturing
protocols.
14. Why does one see a very weak or no
signal in Western blot? ! TOP
There are several possible causes for this. In some
cases, the antibody just recognize a conformational
epitope that ca not be detected in nature condition.
ELISA, immunoprecipitation, or another appropriate
immunochemical assay can be used to confirm the
antibody integrity. In other cases the antibody
may have low affinity. Here one can increase the
incubation period and the concentration of primary
antibody. Weak signal can also result from an inefficient
transfer of antigen from the gel to the membrane.
This is caused by insufficient contact or due to
the presence of air bubbles between the gel and
the membrane or due to an excess of SDS in the gel
or buffer.
15. Too many bands on
a Western blot? ! TOP
It is not uncommon that, contrary to the theoretical
predictions, several bands are detected. Although
it is possible that the antibody is not entirely
specific for the protein, other factors may be responsible:
1. Proteolytic breakdown of the antigen.
This is not uncommon, particularly if samples
are stored for prolonged time or if proteins or
membranes are fractionated after homogenization
of the starting tissue. All additional bands are
of lower apparent molecular mass than the full-length
protein. Particularly susceptible are synapsins
and synaptotagmins. Addition of protease inhibitors
such as PMSF, pepstatin or leupeptin should be considered.
Several vendors offer protease cocktail reagents
for this purpose. It is possible, of course, that
some of the protein was degraded prior to extraction,
in which case it is not possible to remove these
products.
2. Insufficient denaturation and/or reduction.
If the sample buffer does not contain sufficient
SDS and/or reducing agent (DTT, 2-mercaptoethanol),
the protein may not be fully dissociated into its
subunits, reduced or denatured. Hence, extra bands
may appear above the desired protein on the gel.
Boiling the sample in sample buffer immediately
prior to loading can reduce these noncovalent interactions
or disulfide linkages. Use of an irreversible reducing
agent such as TCEP, instead of DTT or beta-mercaptoethanol,
may also be helpful.
3. Too much protein per lane or detection
system too sensitive. Overloading of the
gel is one of the most common reasons for "ghost
bands". Immobilized proteins may provide a concentrated
adsorbtive surface to which certain IgG may bind
nonspecifically. Similarly, such nonspecific binding
may be uncovered when highly sensitive detection
systems such as enhanced chemoluminescence are employed.
A dilution series of the starting material usually
clarifies which of the signals are artefactual.
4. Ineffecient blocking. A variety
of different blocking agents are described in the
literature including nonionic detergents and/or
proteins. Change of the blocking conditions may
remedy the problem.
5. Concentration of antigen too low.
The resolution of SDS-PAGE is limited to 50-100
bands. If the relative concentration of the antigen
of interest is too low (less than 0.2% of total
protein), it may be difficult to detect (for instance,
synaptobrevin/VAMP comigrates with histones in cell
homogenates which interfere with its detection).
Signal enhancement may then lead to the appearance
of artifactual bands. Enrichment of the antigen
by fractionation or by immunoprecipitation should
be considered.
6. Unwanted specific reactivities. Antibodies,
especially polyclonals, will sometimes recognize
proteins in a biological sample with similar sequences
as the antigen, such as those in the same protein
family. Non-affinity-purified polyclonal antibodies
will often recognize other bands due to the exposure
of the animal to various other proteins in its lifetime.
This can be addressed by requesting pre-immune serum
and using this as a comparison.
16. Why do I observe diffuse or moderate
to intense staining of several tissue elements by
IHC? ! TOP
Diffuse staining occurs when the specimen is fixed
for too long, resulting in masking of antigenic
determinants due to aldehyde cross-linking and increased
hydrophobicity of tissue. Use of trypsin or other
proteolytic enzymes will breakdown the cross-linking
and render some tissue antigens reactive. In other
instances, the sections may be too thick or the
specimens may contain crushed or necrotic elements.
This leads to a false negative staining accompanied
by intense background staining. On the other hand,
using an insufficiently diluted primary antibody
leads to an intense staining of specimen or positive
control.
Recombinant Protein Production
1. How many custom proteins have you produced
so far? What is the % of successful productions? ! TOP
We have made more than 150 customer proteins with
successful rate at around 75% (yield at 4mg or more
from 2 liter culture and purity at 75%).
2. What is guaranteed provided at the end
of production? Do we have to pay even if there was
no measurable protein produced or if the protein
is still very dirty after your affinity purification? ! TOP
There is two options in terms of protein production
services: Option 1): 3-5mg purified protein with
75% purity will be GUARANTEED. Otherwise. No payment
will be requested. The charge will be $3,100 for
cloning, protein production, and purification in
E.coli. Option 2): You will get whatever amount
of protein purified from 2 liters of culture with
our best-optimized procedure. But NEITHER YIELD
NOR PURITY WILL BE GUARANTEED. The charge will be
$1,800 for cloning, protein expression, and purification.
3. Is payment due upon delivery, or before
start of the project, or subject to milestones (e.g.
30% upfront, 30% upon proofen production, 30% upon
delivery of purified protein)? ! TOP
We charge subject to milestone with 50% upfront
and 50% upon the completion of the project.
4. Are purification conditions native or
denatured (we prefere native)? ! TOP
Most of them are denatured, although native is not
excluded. If you select option 2), we could try
the native condition with out extra charge.
5. Do you offer a discount for His and
GST tagged versions of the same protein (normally
2 x 1800 $)? ! TOP
If you already have expression vector with His or
GST and no new construction is needed, you will
get $600 off from the original price. But we prefer
to make new construction since we have more confident
in using our own parent vectors for protein yield.
5. Do
you offer a discount for His and GST tagged versions
of the same protein (normally 2 x 1800 $)? ! TOP
In most of cases, the protein will be purified from
inclusion body and the activity has not been tested
although the soluble form can be obtained sometimes.
We could provide refolding service for with extra
charge. Talk to our customer support for detail.
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