Helpful Suggestions

Helpful Suggestions:

1. Use 0.1 to 0.2 micron (small pore) PVDF: You will loose less protein when electroblotting and we are able to perform more ancillary chemistries (reduction and alkylation), CNBr etc.) without loosing significant amounts of protein.

Note: You must use small pore PVDF if expecting internal sequence work!

2. Wash PVDF Thoroughly before staining: Once electroblotted onto small pore PVDF, protein can not be easily washed out by water or water/methanol. After electroblotting you have a "solid phase" protein which can be extensively washed (on a rotary shaker for e.g.) with water, do several changes, then water/methanol (20% methanol for hydrophilic proteins), up to 50% methanol for more hydrophobic proteins, and finally water again. If you are using Tris-Gly buffers you must thoroughly wash before staining to eliminate most Glycine! Residual Glycine will show up in your protein during sequencing and make it impossible to identify actual Glycine for several cycles. If you find it very difficult to de-stain or locate your bands, it may be that there is a lot of residual Glycine still in the PVDF.

3. Electroblot Immediately! : Do not let proteins sit in gels for any length of time as free acrylamide and other ions will adversely effect the protein.

4. Do not over stain! : For most small pore PVDF, protocols call for saturating PVDF in 100% methanol for a few seconds prior to staining. Always use fresh Coomassie staining solution and stain for no longer than 1 minute (45 seconds is usually preferred). Over staining will not compensate for a small amount of protein! Also, rinse the PVDF thoroughly with dd H2O after staining to remove acetic acid -there should be no acetic acid odor !!

5. Dry the PVDF Membrane: After locating bands of interest, carefully excise the band with a sharp razor blade including only the visibly stained PVDF (including empty PVDF will cause the sample to sequence less effectively. Next allow the excised band to dry thoroughly on the bench top at room temperature, typically for 15 to 20 minutes. The band is now ready to ship or store in the freezer. For shipping place the band in a 1.5ml Eppendorf type tube, cover the tube top with parafilm, and send via overnight delivery. (Federal Express or DHL preferred because of excellent tracking methods). There is no need to use dry ice!

6. If you must use Urea !! : Most protocols for 2D gel systems call for using Urea as a chaotrophic agent. Be aware that Urea breaks down quickly (especially at elevated temperatures) to form cyanates which will N-BLOCK proteins! Urea can be successfully used if it is made up fresh just prior to use, being careful to avoid heating the solution (keep the solution below 30 degree's centigrade if possible). Organize your protocol to expose your protein to Urea as briefly as possible -done in the cold if possible. The samples should be electrophoresed immediately and the protein transferred to PVDF immediately!

Technical Questions: E-MAIL: Proseq@tiac.Net

	Proseq, Inc. Protein Sequencing Services \| home Proseq, Inc. Protein Sequencing Services \| About Us \| Sample Requirements \| Helpful Suggestions \| Reasonable Expectations \| Order Form \| New Account Form \| Contact Us \| Title 29
	Helpful Suggestions: 1. Use 0.1 to 0.2 micron (small pore) PVDF: You will loose less protein when electroblotting and we are able to perform more ancillary chemistries (reduction and alkylation), CNBr etc.) without loosing significant amounts of protein. Note: You must use small pore PVDF if expecting internal sequence work! 2. Wash PVDF Thoroughly before staining: Once electroblotted onto small pore PVDF, protein can not be easily washed out by water or water/methanol. After electroblotting you have a "solid phase" protein which can be extensively washed (on a rotary shaker for e.g.) with water, do several changes, then water/methanol (20% methanol for hydrophilic proteins), up to 50% methanol for more hydrophobic proteins, and finally water again. If you are using Tris-Gly buffers you must thoroughly wash before staining to eliminate most Glycine! Residual Glycine will show up in your protein during sequencing and make it impossible to identify actual Glycine for several cycles. If you find it very difficult to de-stain or locate your bands, it may be that there is a lot of residual Glycine still in the PVDF. 3. Electroblot Immediately! : Do not let proteins sit in gels for any length of time as free acrylamide and other ions will adversely effect the protein. 4. Do not over stain! : For most small pore PVDF, protocols call for saturating PVDF in 100% methanol for a few seconds prior to staining. Always use fresh Coomassie staining solution and stain for no longer than 1 minute (45 seconds is usually preferred). Over staining will not compensate for a small amount of protein! Also, rinse the PVDF thoroughly with dd H2O after staining to remove acetic acid -there should be no acetic acid odor !! 5. Dry the PVDF Membrane: After locating bands of interest, carefully excise the band with a sharp razor blade including only the visibly stained PVDF (including empty PVDF will cause the sample to sequence less effectively. Next allow the excised band to dry thoroughly on the bench top at room temperature, typically for 15 to 20 minutes. The band is now ready to ship or store in the freezer. For shipping place the band in a 1.5ml Eppendorf type tube, cover the tube top with parafilm, and send via overnight delivery. (Federal Express or DHL preferred because of excellent tracking methods). There is no need to use dry ice! 6. If you must use Urea !! : Most protocols for 2D gel systems call for using Urea as a chaotrophic agent. Be aware that Urea breaks down quickly (especially at elevated temperatures) to form cyanates which will N-BLOCK proteins! Urea can be successfully used if it is made up fresh just prior to use, being careful to avoid heating the solution (keep the solution below 30 degree's centigrade if possible). Organize your protocol to expose your protein to Urea as briefly as possible -done in the cold if possible. The samples should be electrophoresed immediately and the protein transferred to PVDF immediately! Technical Questions: E-MAIL: Proseq@tiac.Net