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RIPA Lysis and Extraction Buffer, 250 mL - FAQs

RIPA Lysis and Extraction Buffer, 250 mL - FAQs

View additional product information for RIPA Lysis and Extraction Buffer - FAQs (89901, 89900)

8 product FAQs found

What are the standard lysis buffers used with mammalian cells for detection of protein expression by immunoprecipitation (IP) or Western blot analysis?

The most commonly used buffer is RIPA Buffer with SDS. We offer RIPA Buffer (Cat. Nos. 89900 and 89901). We also offer the Pierce IP Lysis buffer (Cat. Nos. 87787 and 87788) as well as M-PER (Cat. Nos. 78501, 78503, and 78505).

Find additional tips, troubleshooting help, and resources within our Cell Lysis and Fractionation Support Center.

Does RIPA Lysis and Extraction Buffer (Cat. No. 89900, 89901) contain protease or phosphatase inhibitors?

RIPA Lysis and Extraction Buffer (Cat. No. 89900, 89901) does not contain protease or phosphatase inhibitors. If desired, you may add protease and/or phosphatase inhibitors, such as Halt Protease Inhibitor Cocktail (Cat. No. 78410) and Halt Phosphatase Inhibitor Cocktail (Cat. No. 78420) to the RIPA Lysis and Extraction Buffer to prevent proteolysis and maintain phosphorylation status of proteins. We recommend adding protease and phosphatase inhibitors immediately before use.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Why is it not recommended that I use RIPA buffer for protein A280 measurements with my NanoDrop spectrophotometer?

RIPA buffer produces a particularly strong absorbance signal at the 280 nm wavelength. As a result, it will either over estimate or under estimate protein concentrations and interfere with the protein purity ratio.
Protein samples in RIPA buffer should be quantified via the Pierce Protein 660 or BCA colorimetric assays using a full spectrum NanoDrop model.

Find additional tips, troubleshooting help, and resources within ourProtein Purification and Isolation Support Center.

Why is there low phosphorylation of the proteins when I use the RIPA Lysis and Extraction Buffer?

Low phosphorylation is usually due to phosphatase activity. We recommend adding a Halt Phosphatase Inhibitor Cocktail to the buffer before use.
Alternatively, the protein is not phosphorylated or phosphorylated at a low level.

Find additional tips, troubleshooting help, and resources within our Cell Lysis and Fractionation Support Center.

What if proteolysis occurs when I use RIPA Lysis and Extraction Buffer?

Proteolysis indicates that no protease inhibitors were added. We recommend adding a Halt Protease Inhibitor Cocktail to the RIPA Lysis and Extraction Buffer before use.

Find additional tips, troubleshooting help, and resources within ourProtein Purification and Isolation Support Center.

I am getting a low concentration of proteins when using the RIPA Lysis and Extraction Buffer. What could be a possible cause and solution?

Most likely excess buffer was used. Use less buffer (e.g., 0.25 mL to 0.5 mL per 75cm^2 flask containing 5 x 10^6 cells), but use a sufficient amount to cover the entire plate.

Find additional tips, troubleshooting help, and resources within our Cell Lysis and Fractionation Support Center.

Why is the total protein yield low when I use the RIPA Lysis and Extraction Buffer?

Some cells are more resistant to lysis than others and can cause a low protein yield. Make sure the cell pellet is thoroughly suspended in RIPA Buffer, incubate for longer with occasional swirling, and sonicate the pellet to increase yield.

Find additional tips, troubleshooting help, and resources within our Cell Lysis and Fractionation Support Center.

What lysis buffers do you offer for mammalian cells?

Please see the various lysis buffers we offer (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/cell-lysis-total-protein-extraction.html) for total protein extraction from mammalian cells.

Find additional tips, troubleshooting help, and resources within our Cell Lysis and Fractionation Support Center.