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Pierce™ High pH Reversed-Phase Peptide Fractionation Kit - FAQs

Pierce™ High pH Reversed-Phase Peptide Fractionation Kit - FAQs

View additional product information for Pierce™ High pH Reversed-Phase Peptide Fractionation Kit - FAQs (84868)

6 product FAQs found

Can I purchase the Reversed-Phase Fractionation Spin Columns from the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868) separately?

No, the spin columns from this kit cannot be purchased separately. However, if you are only using these columns to remove salts from your peptide samples, you can purchase the Pierce Peptide Desalting Spin Columns (Cat. No. 89851, 89852), as an alternative.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

How much starting material is needed to fractionate my samples using the Pierce High pH Reversed Phase Fractionation Kit (Cat. No. 84868)?

A minimum of 100 µg of protein as starting material from the digestion step is needed for fractionation using eight fractions. Less starting material would be needed if fewer fractions are used. If peptides are being measured, the range of peptide to be loaded on these columns is 10 to 100 µg.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

I want to fractionate a complex mass spectrometry sample, but my sample is different from the one described in the manual for the Pierce High pH Reversed-Phase Peptide Fractionation Kit. Should I use a customized fractionation gradient?

Recommended gradients are available for typtic protein digest samples with or without TMT labeling. Use of other chemical labels, performing selective peptide enrichment, or using a different digestion enzyme other than trypsin may affect the elution profile of the peptides during fractionation. For the best sample peptide coverage, an ideal elution profile would result in relatively equal amounts of peptides in each fraction.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

After running the Pierce TMT11plex Yeast Digest Standard, I observe low signal to noise ratio and poor quantitation accuracy for the Tandem Mass Tag (TMT) reporter ions. What do you recommend?

Here are some recommendations:

- Clean and recalibrate the mass spectrometry instrument using one of our Pierce Calibration Solutions (https://www.thermofisher.com/search/browse/category/us/en/600654).
- Verify settings for liquid chromatography (LC) acquisition methods.
- Fractionate TMT-labeled samples using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868) to reduce sample complexity.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

I have a lower number of peptide/protein identification in my combined TMT-labeled samples compared to the unlabeled sample. What can I do?

We recommend fractionating the sample using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868), to reduce the sample complexity before LC-MS analysis and thus increase the number of quantifiable peptides/proteins for multiplexed samples.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

After TMT reagent labeling, I still see a lot of excess TMT tags in my sample. How should I remove it?

We offer EasyPep MS sample preparation kits (EasyPep Maxi Sample Prep Kit (Cat. No. A45734), EasyPep Mini MS Sample Prep Kit (Cat. No. A40006), EasyPep 96 MS Sample Prep Kit (Cat. No. A45733)) that contain wash buffers specifically formulated to clean up TMT-labeled protein digests. To remove excess TMT reagent from samples prepared using other sample preparation methods, we recommend using Pierce Peptide Desalting Spin Columns (Cat. Nos. 89851, 89852) with extra washes using 5% methanol. The Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868) can also be used to remove excess unreacted TMT tags before collecting fractions.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.