Product FAQ

I stored my diluted antibody in the refrigerator for a week, and now it doesn’t work. What happened?

Answer

In all likelihood, your antibody stopped working because it lost its reactivity. This happens because antibodies (and most other proteins) are less stable at low concentrations (e.g., the μg/mL range and lower) than they are at higher concentrations. For example, proteins adsorb to surfaces like the walls of their containers due to charge-mediated and hydrophobic interactions. This occurs regardless of the protein concentration, and it usually results in some degree of protein denaturation and loss of activity. However, at low protein concentrations the impact of adsorption is larger per unit of time than at higher concentrations. Antibodies in solution also aggregate with each other for the same reasons that they adsorb to surfaces, sometimes resulting in loss of activity. How fast your diluted antibody loses activity in storage is unpredictable, so store your diluted antibodies no longer than overnight at 2-8 degrees C and then discard them. Better yet, make a fresh working dilution each time you need to use the antibody.

Answer Id: E12605

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Product FAQ

Why doesn’t my antibody recognize the native protein in my application?

Answer

Antibodies prepared against a short peptide sequence may not always recognize the full-length protein in which that peptide sequence is located. The peptide sequence only represents a small portion of the entire protein, and the full-length protein is usually a more complex structure with folds, alpha-helices, beta-sheets, and other structural motifs as well as various posttranslational modifications, any of which can shield the epitope from the antibody. This is one reason it is important to read the manuals for our antibodies. They describe the capabilities and specificity of our antibodies and list the applications for which these products are validated.

Answer Id: E12606

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Product FAQ

What options do you offer for performing ELISAs?

Answer

We offer full ELISA kits, Reagent Sets and Antibody Pairs. ELISA kits are complete, ready-to-use kits with pre-coated plates, all buffers, capture, and detection antibodies included. Most kits are single plate format, but some are available in 2- or 5-plate formats. Reagent Sets are for researchers who need the core kit components but prefer to make their own buffers and coat their own plates. Antibody Pairs are matched pairs of detection and capture antibodies for researchers who need to process large numbers of samples.

Answer Id: E12607

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Product FAQ

What is the excitation/emission of the RedoxSensor™ Green reagent?

Answer

The approximate excitation/emission of the RedoxSensor™ Green reagent, in reduced form, is 490/520 nm.

Answer Id: E10102

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Product FAQ

How do your phosphospecific ELISA kits compare to immunoprecipitation and western blotting?

Answer

Our phosphospecific ELISA kits have several advantages, including ease of use and increased sensitivity. Phosphospecific ELISA kits are typically 2-10 times more sensitive than western blots, so they are particularly useful for the detection of “low-expressing” proteins or for small sample sizes. In addition, with the use of the recombinant standards provided in the kit, phosphospecific ELISAs provide quantitative results without having to perform densitometry.

Answer Id: E12624

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Product FAQ

What are the different types of ELISA kits you offer?

Answer

Our ELISA kits can be categorized into several different groups (see Table 2, Page 35 of the Protein Analysis Handbook - http://www.thermofisher.com/us/en/home/global/forms/protein-handbook-registration.html), based on a number of factors: target protein class, sensitivity, readout method, or ability to detect specific phosphorylation states of the target protein.

Standard colorimetric ELISA kits use a standard colorimetric readout and allow excellent sensitivity and detection range. Typical sensitivity is less than 10 pg/mL and standard curve range is around 10-250 pg/mL, although there are some kits with wider ranges.
Ultrasensitive ELISA kits use a standard colorimetric readout but enable detection and analysis of proteins to levels as low as 0.5 pg/mL. With measurement range of 0.5-20 pg/mL, these kits are especially useful with highly diluted samples.
Chemi ELISA kits use chemiluminescence detection for high sensitivity (less than 1 pg/mL) and are highly flexible with a wide measurement range from 0.5 to 2000 pg/mL.
Phospho ELISA kits enable the specific detection of phosphorylation of key signaling proteins with high specificity, and are often used to supplement western blot results and provide quantitative data.

Answer Id: E12608

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Product FAQ

What transfection reagent should I use for transfecting siRNA?

Answer

The optimal transfection agent depends upon the cell type used. That being said, we find that Lipofectamine™ RNAiMAX transfection agent provides excellent transfection efficiency with low toxicity in most cell types.

Answer Id: E6585

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Product FAQ

Can I wash my SYPRO™ Ruby stained blots longer than the recommended 4-6 times/1 min in deionized water for nitrocellulose membranes or 2-3 times/1 min for PVDF membranes?

Answer

Yes. Blots can be left in water indefinitely without destaining the stained proteins. Longer water washes can help to reduce high non-specific background, especially for immersion-stained PVDF membranes.

Answer Id: E11154

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Product FAQ

Can I use the lentiviral expression vector by itself as an expression vector (without the packaging mix)?

Answer

Yes, the lentiviral expression vector will work as an expression vector by itself and can be stably selected with the appropriate antibiotic. Please note that the vector will be about twice the size of most regular vectors. Therefore, you may need to increase the amount of transfected vector to approximate molar equivalents.

Answer Id: E9294

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Product FAQ

Why do bands from DNA ladders cross-hybridize with some probes?

Answer

Some bands in DNA ladders are generated from plasmids similar to pUC.

Answer Id: E3092

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Product FAQ

I'm seeing no to low DNA yield when using the GMO extraction kit. What should I do?

Answer

No to low yield can be due to incomplete lysis of the sample, incorrect preparation of reagents, or non-optimal elution of the DNA from the column.

Answer Id: E10103

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Product FAQ

How should I adjust the Platinum™ Pfx DNA Polymerase protocol if I am trying to generate an amplicon greater than 2 kb, or if I am starting with long PCR primers?

Answer

To generate amplicons greater than 2 kb, use 2.5 units of Platinum™ Pfx polymerase (instead of 1 unit), decrease the extension temperature to 68 degrees C, and increase the extension time to 1 kb/minute.

If long PCR primers are used with Platinum™ Pfx polymerase, increase the magnesium concentration in the reaction to 1.5 mM.

Answer Id: E3053

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Product FAQ

What are the fluorescence excitation/emission maxima for the dyes contained in the Chromatin Condensation/Dead Cell Apoptosis Kit with Hoechst 33342 and PI for Flow Cytometry aka Vybrant™ Apoptosis Assay Kit #5, Hoechst 33342/Propidium Iodide)?

Answer

The excitation/emission maxima for the 2 dyes in the kit are shown below:

Hoechst 33342: 350/461 nm, bound to DNA
Propidium iodide: 535/617 nm, bound to DNA

Answer Id: E9831

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Product FAQ

I was able to see a signal on my blots with a WesternDot™ conjugate originally, but the signal is gone after a few days. What happened?

Answer

WesternDot™ signals can fade when bound to dried PVDF membranes. For archiving stained blots or imaging after more than 24 hours, we recommend using nitrocellulose membranes.

Answer Id: E11324

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Product FAQ

My sample lysates were made in RIPA buffer. Can I test these samples with your ELISA kits?

Answer

Yes, you can. The composition of the traditional 1X RIPA buffer is very similar to that of our Cell Extraction Buffer (Cat. No. FNN0011). Cat. No. FNN0011 is frequently used to prepare lysates for testing with our ELISA and Luminex™ kits. Our NP-40-based Cell Extraction Buffer (Cat. No. FNN0021) is also used. We recommend diluting lysates made with Cat. No. FNN0011 at least 10-fold in order to lower the SDS concentration to less than or equal to 0.01% (v/v) before adding the samples to the ELISA or Luminex™ assay.

Answer Id: E12625

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