I am preparing cell membranes for 35s GTP gamma assay, I have two samples of 1.66 mg/ml and 1.4mg/ml protein concentration , and both are 100 microliters volume, how can I dilute them to be kept in freezer?
Sergio Grunbaum
Determine the desired final protein concentration: Decide on the target protein concentration you want to achieve after dilution. This will depend on the assay requirements and the concentration range within which the assay can accurately detect GTP gamma binding. Calculate the dilution factor: To calculate the dilution factor, divide the desired final protein concentration by the current protein concentration. For example, if you want to achieve a final protein concentration of 0.5 mg/ml and you have a sample with a protein concentration of 1.66 mg/ml, the dilution factor would be 1.66 mg/ml / 0.5 mg/ml = 3.32. Prepare dilution buffer: Prepare a suitable dilution buffer that is compatible with the 35sGTP gamma assay and your membrane samples. The dilution buffer should maintain the stability and activity of the proteins while providing the necessary conditions for the assay. Commonly used buffers include Tris-HCl or phosphate buffer, but the specific buffer choice will depend on the assay requirements. Dilute the samples: Take an appropriate volume of each sample to achieve the desired dilution. For example, if you have 100 μl of a 1.66 mg/ml sample and want to achieve a final concentration of 0.5 mg/ml, you would take 100 μl of the sample and add 200 μl of dilution buffer to achieve a total volume of 300 μl. Mix the solution thoroughly to ensure homogeneity. Prepare aliquots for storage: Divide the diluted samples into smaller aliquots to facilitate storage and avoid repeated freeze-thaw cycles, which can affect protein stability. The size of the aliquots will depend on your future experimental needs, but common volumes range from 50 μl to 200 μl. Label the aliquots with the sample name, dilution factor, and date. Freeze the aliquots: Place the aliquots in suitable storage containers, such as cryovials, and store them in a freezer at a temperature appropriate for the stability of your membrane proteins. Typically, -20°C or -80°C are commonly used temperatures for long-term storage.
To dilute your crude membrane samples for the 35sGTP gamma assay and prepare them for storage in the freezer, you can follow these steps:
By following these steps, you should be able to dilute your crude membrane samples for the 35sGTP gamma assay and store them appropriately for future use.
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Aisha Abdelmalik
Thanks.
after diluting them to the appropriate concentration of 1mg/ml. And freezed as 100microliter aliqout.
the amount required for the assay is 10 microgram per well, do I have ti dilute them more in the assay buffer?
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Sergio Grunbaum
Determine the current protein concentration of your crude membrane samples. Let's assume you have Sample A with a concentration of X mg/ml and Sample B with a concentration of Y mg/ml. Calculate the dilution factor needed to achieve a final concentration of 1 mg/ml for both samples. Divide the current concentration (X or Y) by 1 mg/ml to obtain the dilution factor. For example, if Sample A has a concentration of 1.66 mg/ml, the dilution factor is 1.66/1 = 1.66. Prepare a dilution buffer suitable for your assay. This buffer should maintain the stability and activity of your samples. Use a buffer that is commonly used for membrane protein assays or consult your assay protocol for specific recommendations. Calculate the volume of dilution buffer required to achieve a 1 mg/ml concentration for each sample. Divide the desired final volume (100 μl) by the dilution factor obtained in step 2. For example, if the dilution factor for Sample A is 1.66, the volume of dilution buffer needed is 100 μl/1.66 = 60.24 μl. Add the calculated volume of dilution buffer to each sample and mix gently to ensure proper dilution. Aliquot 100 μl of each diluted sample into separate tubes suitable for freezing. Label the tubes with sample identifiers and the concentration (1 mg/ml). Freeze the aliquots at the appropriate temperature (-20°C or -80°C) for storage.
By following these steps, you will have diluted your crude membrane samples to a concentration of 1 mg/ml and stored them as 100 μl aliquots for future use in the 35sGTP gamma assay.
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