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Technical FAQs
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Dear Customer,

Below is a list of different technical questions ranging from product content to product storage. Do not hesitate to contact our headquarters at techsupport@cohesionbio.com and your inquiry will gladly be forwarded to the appropriate department.


Are your products tested for endotoxin levels?

None of our products are specified as endotoxin-free.


How to properly handle a peptide vial?

Before opening, the vial should be allowed to reach ambient temperature. Before opening the vial, and unless stated otherwise, it is strongly recommended to centrifuge all product preparations before use (10000 x g 5 min) to drive any peptide that may be lodged in the cap or on the side to the bottom of the vial. The peptide has to be weighed out quickly and the vial resealed. Reconstituting the entire sample and aliquoting the resulting solution as desired is preferable. It is also recommended to avoid thawing/freezing cycles, which tend to degrade the peptide.


What is the blocking peptide and how is it used?

The blocking peptide is the antigen used for the immunization of the rabbits in order to obtain the antigen-specific antibody. The blocking peptide can be used as a negative control in a western blot in order to verify the specificity of the bands detected with the antibody.

The use of the blocking peptide in brief is as follows:

Incubate the antibody, in parallel, with and without the blocking peptide (the ratio antibody/blocking peptide is available in the certificate of analysis delivered with the antibody) in a small volume (500ul of 1%BSA in PBS or 3% skim milk) for 1 hour in room temperature with rotation. After the incubation time, dilute each vial to the desired working dilution with the buffer you use and apply it to the membranes for parallel experiments.

Compare the results of antibody alone versus antibody with blocking peptide. The disappearance of the requested band will verify the specificity of the antibody.


How should peptides be stored?

For long-term storage the peptides should be kept in solid form at < -20°C. For shorter storage, +4°C will suffice. Dissolved peptides are less stable than the lyophilized form. We recommend aliquoting the solutions before freezing in order to avoid thawing/freezing cycles, which tend to degrade the peptides. The stability of the aliquots may range from weeks to months.


How to properly handle an antibody vial?

Before opening, the vial should be allowed to reach ambient temperature. Before opening the vial, and unless stated otherwise, it is strongly recommended to centrifuge all product preparations before use (10000 x g 5 min) to drive any antibody that may be lodged in the cap or on the side to the bottom of the vial. It is also recommended to avoid thawing/freezing cycles, which tend to degrade the peptide/antibody.


How should antibodies be stored?

For long-term storage the antibodies should be kept in solid form at < -20°C. For shorter storage, +4°C will suffice. Dissolved peptides are less stable than the lyophilized form. We recommend aliquoting the solutions before freezing in order to avoid thawing/freezing cycles, which tend to degrade the antibodies. The stability of the aliquots may range from weeks to months.


Why do different lots of the same antibody give different western blot results?

Cohesion Biosciences some antibodies are polyclonal. Therefore, differences between different batches may occur. Our protocols are designed to minimize these differences but we cannot completely avoid them. Each lot is purified by affinity column, using identical protocols and conditions. The antibodies undergo quality control using western blot and are released only after satisfactory results are obtained in comparison with the previous lot. Nevertheless, the concentration and the conditions of use (dilution, primary antibody incubation time, exposure time etc.) can change between different batches and may require further calibration.


Why are there differences between the detected MW band in a western blot and the calculated one?

The calculated MW of a protein is based only by its amino acid sequence and does not take into account any posttranslational modifications such as glycosylation, phosphorylation and others. Therefore, in a western blot you might see the expected MW and/or a modification of the protein that runs with a different MW.


How do you avoid cross reactivity between close members of the same protein family?

Cohesion Biosciences puts great effort in producing highly specific antibodies.

We carefully choose the epitope for immunization, taking into account two main parameters:

a. Immunogenecity

b. Specificity of the epitope.

Before immunization, we run a search to verify that the chosen epitope has maximum homology with other species and minimum homology among members of the same family, or other proteins.

The antibodies are affinity purified on immobilized antigens (the injected peptides).

All our antibodies undergo strict quality control by western blot analysis including the use of blocking peptide as negative control to verify the specificity of the antibody. Cohesion Biosciences supplies the blocking peptide, so that every customer can check the specificity of the antibody with its own system.

Each antibody is supplied with a specific datasheet describing the product and its quality control results, in the presence or absence of the control antigen.


How long does a project take for Custom Polyclonal Antibody?

Polyclonal antibody production requires 3-4 months depending on antigen and length of immunization procedure. Usually, the process is faster when using a protein than for a peptide. If the animal does not respond to the immunization, more boosts are planned before blood collection.


What kind of antigens do you use to make antibodies?

Cohesion Biosciences’ protocols use peptides to make antibodies. The antigen is conjugated to a carrier protein such as KLH to make the antigen immunogenic. For peptide antigens, we recommend adding a cysteine residue at the C-terminus end of the peptide sequence to allow for efficient conjugation using the free sulhydryl chemical group.


Can Cohesion Biosciences help me design a good peptide sequence for my antigen?

Cohesion Biosciences technical staff is highly experienced in antibody development and epitope design. Whereas most companies rely on bioinformatic algorithms, Cohesion Biosciences staff is able to take into account the protein surface available for antibody binding by using a 3D protein design software. Ultimately, the peptide candidates are reviewed by a qualified Ph.D. scientist before the final recommendations are sent to the client. This service is free of charge when Cohesion Biosciences synthesizes the peptide. Contact our Technical Support Dept to send your protein sequence or corresponding protein accession number.


How much antigen is required for Custom Polyclonal Antibody?

For animal immunization, 10 mg of a peptide is required. An additional 5 mg is needed when antigen-affinity purification is performed. The peptide should be supplied in a solid format free of toxic additives such as PMSF or sodium azide. When a protein is used as antigen, 5 mg is sufficient. When the protein is in solution, the concentration should be at 1 mg/ml in a buffer solution free of detergents. When the protein is insoluble, using an aggregate is the best format instead of a solubilization buffer containing denaturing agents.


Can you guarantee that for the Custom Polyclonal Antibody will work for me?

Cohesion Biosciences offers you the best recommendations and antibody production experience to minimize the chances to fail at making the requested antibody. There is no guarantee that the antibody that Cohesion Biosciences produces for you will work for your application. Some projects are at higher risks than others and Cohesion Biosciences will inform you of the chance of failure at project initiation. Cohesion Biosciences still guarantees that the antibody generated using an antigen synthesized by Cohesion Biosciences will recognize the antigen in a direct ELISA.


What is the average turnaround time for peptide synthesis?

Although most peptides can be shipped out within 3-4 weeks, actual processing time is dependent on the peptide sequence, length, purity and modifications. Please consult with our Technical Support Dept for an estimated timeline of the peptide synthesis process. Estimated delivery times are stated on all quotations.


How is siRNA synthesized?

The most commonly used method in siRNA synthesizers is the 'phosphite triester' method, developed by Koster. This method builds the phosphodiester backbone of the RNA molecule by using β-cyanoethyl phosphoramidites. The synthesis begins with a solid support (CPG) with an attached starter nucleoside. A deblocking-coupling-capping-oxidation cycle is repeated to reach the desired length of siRNA molecule. After synthesis, ammonia is used to detach the siRNA from the solid support. The siRNA then undergoes deprotection and TBDMS treatment before being purified. The end result of these processes is pure siRNA. Each synthesized ss-RNA is annealed to produce the final duplexed siRNA form. 


What information we need to provide for the synthesis of siRNA? 

You need to provide 19 or more nucleotides sequence with dTdT, or you can provide the related Gene ID or Accession Number for free design. Please fill products information and delivery information in Custom siRNA Synthesis Online Quotation Request Form.


Can Cohesion Biosciences synthesize chimeric RNA?

Yes, we can. We have the ability to synthesize chimeric RNA molecules that incorporate dA, dC, dG, and dT DNA bases. We can also incorporate 2'-O-Methyl and 2'-F(rU, rC) into the RNA. 


Are phosphate groups present on the 5' or 3' ends of the synthesized siRNA?

Unless explicitly stated, the 5' and 3' ends are capped with -OH groups. Therefore, to order 5' phosphate-capped siRNAs, you must request for 5' phosphorylation modification. 


What types of modified siRNAs are there?

In order to maximize siRNA applications, modified siRNAs are available. Although there are many ways to modify siRNAs, the most convenient method is to use phosphoramidites to label siRNAs during synthesis. For a 5' siRNA modification, synthesis is first conducted as normal. At the final stage, however, a labeling phosphoramidite is attached to the 5' end, resulting in a 5' labeled siRNA. 3' labeling is somewhat complicated in that we must start with a labeled phosphoramidite attached to a solid support and build the molecule from there. You can incorporate the label of your choice to the siRNA sequence by using a labeled phosphoramidite at the 5' end of deoxyuridine, therefore allowing for an internal modified siRNA. 

The following modifications are offered for our siRNAs.

5' Amine - 3' amine modification

5' Phosphorylation phosphorylation

5' Thiol Modification

5' Biotin Modification

5' Cholesterol - 3' Cholesterol modification

5' Fam - 3' Fam modification

5' TET modification

5' Cy3 modification 

F-rU, F-rC Internal modification

OMe-rA, OMe-rG, OMe-rC, OMe-rU Internal modification


How to I store my siRNAs and how long do they keep?

siRNAs can normally be kept stable at -20°C for very long time. The lyophilized form is especially stable and has a long shelf-life. Although solution-dissolved siRNAs can be stable, contamination of the reconstitution solution with RNase will degrade the product. Also, repeated freeze-thaw cycles accelerate the degradation process. Therefore, we recommend that after you receive the siRNA stock, you reconstitute and make several aliquots to avoid such freeze-thawing. Because the phosphodiester bonds of the RNA can break under high pH conditions, we ask you to take caution, and recommend reconstituting in ultrapure water provided. 


How do I reconstitute my siRNA?

If you would like to keep your siRNA in solution, we recommend reconstituting with DEPC-treated water that we provide with your order to maximize stability. 


How to I store my fluorescent dye modified siRNA?

Photobleaching may occur if the fluorescent dye modified siRNA is exposed to light for prolonged periods of time. Therefore we recommend that you store such siRNAs in a dark container and store that container in a dark place.


What does the siRNA O.D. value mean?

Because the amount of synthesized siRNA is extremely small, it is very difficult to quantify by weight. Therefore, we capitalize on the light-absorption properties of nucleic acids to indirectly measure the amount of product. The amount of light absorbed by a given amount of product is expressed as an O.D. (Optical Density) value, and is directly related to the amount of product in solution. 


How do I calculate the amount of siRNA from the O.D. value?

After measuring the amount of 260 nm light absorbed by the siRNA, the following formula is used to calculate the actual amount of siRNA O.D. = εC. The ε is the extinction coefficient which is unique for different materials, and the C stands for the siRNA concentration. If one knows the extinction coefficient and the O.D. value of the siRNA, the concentration can be calculated by substituting those values in the formula above. The ε value for an siRNA molecule is the sum of the extinction coefficient of each base constituting the molecule. The extinction coefficients for each base at 260 nm is are as follows: 

rG : 11.5 ml/μmole

rC : 7.2 ml/μmole

rA : 15.4 ml/μmole

rU : 9.9 ml/μmole 

Therefore, the extinction coefficient for the entire siRNA molecule can be calculated by multiplying the number of bases by the corresponding extinction coefficient and adding all four products together. 


If my 18-mer siRNA (3G, 4C, 5A, 6U) O.D. value was 0.7, then how much siRNA do I have?

Let's start by calculating the extinction coefficient (εε) of the entire siRNA molecule.

ε= 11.5 x 3 + 7.2 x 4 + 15.4 x 5 + 9.9 x 6 = 199.7 (ml/μmole)

Therefore, using the O.D. = εC formula, we can calculate the final concentration C to be C = 0.7 /199.7 = 0.0035 (μmole/ml) = 3.5 (nmole/ml). 


How do I calculate the molecular weight of the synthesized siRNA molecule?

Substitute the number of each base into the following formula: M.W. = (NA x 329.208) + (NC x 305.183) + (NG x 345.207) + (NU x 306.168) - 63.98) + 2.016 NA = Total number of rA NC = Total number of rC NG = Total number of rG NU = Total number of rU 


Can I know how many ng the synthesized product is?

Normally, we will fulfill an order with a guaranteed nmole amount, and the synthesis report will also report the final amount in nmoles. If you must have the ng amount to calculate for an experiment, you can convert from nmole to ng by using the formula below. We make it easy for you by giving you the molecular weight of the siRNA sequence in the report. Molecular Weight (g) X mole (nmole) = Mass of siRNA (ng)


How do I reach a target concentration?

Each synthesis report gives you a 'volume for μM' value that stands for the volume of DEPC-treated water you need to add to achieve a concentration. As an example, if the 'volume for 100μM ' values for two siRNAs are 100 and 200, then you can reconstitute with 100μl and 200 μl of DEPC-treated water, respectively, to reach a 100μM concentration.

In other words, the siRNA contained in each tube is:

100 μl x 100μM = 10,000 pmole = 10 nmole,

200 μl x 100μM = 20,000 pmole = 20 nmole

Remark: 

1 pmole/μl = 1x10-12 mole / 1x10-6 L = 1x10-6 mole/L = 1 μmole/L = 1 μM


How do I convert between morlarity and moles?

The standard concentration units for oligomers is given in M (mole/L), and the prefixes such as μ (micro), n (nano), p (pico) etc. describe the scope of the unit. The following prefixes are used not only for M, but for other measurement units such as length, mass etc. 

10-1 = deci [d] 101 = deca [da]

10-2 = centi [c] 102 = hecto [h]

10-3 = milli [m] 103 = kilo [k]

10-6 = micro [μ] 106 = mega [M]

10-9 = nano [n] 109 = giga [G]

10-12 = pico [p] 1012 = tera [T]

10-15 = femto [f] 1015 = peta [P]

10-18 = atto [a] 1018 = exa [E]

10-21 = zepto [z] 1021 = zetta [Z]

10-24 = yocto [y] 1024 = yotta [Y] 

1 pmole/μl = 1x10-12 mole / 1x10-6 L = 1x10-6 mole/L = 1 μmole/L = 1 μM 

Therefore, μM and pmole/μl are one and the same.

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