User manual / Technical Information
Contents
1. iStructions for use Roti Mark 10 150 Protein molecular weight marker for SDS PAGE kD 150 100 80 60 40 30 20 ar 10 Figure 4 20 gradient gel Tris SDS Carl Roth GmbH Co KG SchoemperlenstraBe 3 5 76185 Karlsruhe Postfach 100121 76231 Karlsruhe Telefon 49 0 721 5606 0 Telefax 49 0 721 5606 149 E Mail info carlroth de Internet www carlroth de e d c w 02 2015 I Introduction Roti Mark 10 150 is composed of a set of genetically engineered in vitro expressed proteins with defined regular molecular weights which stand out for their homogeneous level running properties see Table 1 This makes it easier to visually estimate the apparent molecular weight of your protein For easier orientation in the gel the 40 kD band shows a stronger intensity Proteins have been adjusted to equal apparent masses after Coomassie staining Nearly all bands have a concentration of 50 100 ug ml per band The 40 kD protein has been enhanced to 150 200 ug ml batch specific Roti Mark 10 150 has been assembled from a set of His tagged recombinant proteins that may be detected during Western Blot analysis using anti His antibodies Proteins are pre reduced acylated and dissolved in non reducing Lammli buffer with 0 01 bromophenol blue Tab 1 MW kD logMW 150 5 1761 100 5 002. 00 80 4 9031 60 4 7782 40 4 6021 30 4 4771 20 4 3010 10 4 0000 In top V you will find a simple procedure for determining the apparent molecular weight of a protein in SDS PAGE by comparing it to the running distance of marker proteins ll Storage e The marker will not be shipped cooled or with dry ice This does not affect usability e Please store Roti Mark 10 150 at 20 C The marker can be stored at 4 C for a short period a few days To avoid frequent freezing and thawing aliquots should be frozen e If necessary Roti Mark 10 150 PLUS can be heated slighty before use to resolubilise precipitated SDS e The marker should not be heated to more than 65 C nor stored for a longer period at temperatures above freezing point lll Gel loading e Recommended loading amount for mini gels 10 0 75 mm thick Coomassie staining approx 5 ul Silver staining approx 1 ul e Please note Loading amount required varies depending on gel thickness C T ratio the staining used and width of comb tooth e The intensity of Coomassie staining can turn out very differently depending on the protocol used Two methods which guarantee efficient staining can be found in top VI IV Trouble Shooting Marker bands cannot can only be seen very weakly e Please ensure the correct loading amount The recommended quantity is valid for mini gels with a thickness of 0 75 mm If thicker or larger gels are used the loading amoun
3. all running distance 2 Plot log MW from Table 1 in a graph against the RF values of the marker example see graph LogMW 5 50000 5 30000 5 10000 4 90000 4 70000 4 50000 4 30000 4 10000 3 90000 0 0 2 0 4 0 6 0 8 1 RF values 3 Calculate the RF value of your protein Determine the corresponding logMW by using your graph Calculate the molecular weight in kD according to Table 1 TIP Roti Mark 10 150 is composed of genetically engineered multimeres of a protein and exhibits level running properties In contrast to this irregular running properties appear with marker compounds of varying proteins e g myosin B galactosidase BSA carbonic anhydrase etc Derived molecular weights of unknown proteins can therefore vary depending on the marker used Conformation of the values detected with Roti gt Mark 10 150 to the traditional marker can be achieved with following formula MW irag MW detected 1 471 5 X In MW detected 6 61 9 VI Coomassie staining With Rot Blue Art No A152 Incubate gel 2 to 12 h with Roti Blue as per instructions Decoloring is not necessary With Brilliant Blue G250 Art No 9598 Incubate gel for 30 60 min in fixative under gentle shaking Incubate gel for 20 40 min in staining solution under gentle shaking Incubate gel for 30 sec in fixative under gentle shaking Incubate gel in decoloring solution under gentle shaking until background staini
4. ng has been removed and proteins are clearly visible Fixative 40 ethanol 10 acetic acid Staining solution Mix 50 ml solution and 50 ml solution II directly before use Solution 0 2 Brilliant Blue G250 90 ethanol Solution Il 20 acetic acid Decoloring solution 20 ethanol 10 acetic acid VII Recommended Reagents e Brilliant Blue G250 Art No 9598 e Ethanol p a Art No 9065 e Acetic acid p a Art No 3738 e Roti Blue Art No A152 lt gt Warning H319 P305 P351 P338 P337 P313 Roti Mark 10 150 T850 2 100 ul T850 1 500 ul
5. t must be increased e Improve staining The loading quantity of Roti Mark was optimised to obtain particularly sharp bands and optimal running behaviour Do not try to compensate weak Staining by increasing the protein load This will result in a change of the running behaviour of the proteins of your sample as well as of the marker and in indistinct and thick bands e Few weak marker bands Under certain conditions marker proteins may agglutinate Resolubilise marker aliquots by incubating for 5 min at 65 C Mix carefully Marker bands are fuzzy e Avoid overloading the gel e Please ensure that the marker is not stored at room temperature for a longer period Place the marker on ice between two gel runs e Avoid frequent freezing thawing of marker e Long term storage should always take place at 20 C e Please take care that the gel contains no air bubbles when casting e When casting the gel please ensure that the acrylamide solution is mixed thoroughly e Only use high quality acrylamide solutions e g Rotiphorese Gel 30 Art No 3029 or Gel 40 Art No 3030 e Avoid overheating the gel Reduce voltage if required e Check the composition and pH value of the buffer used V Apparent MW of a protein 1 Determine the RF values of the marker proteins after electrophoresis Start Border of collecting separating gel Front of run Bromophenol blue band Running distance of protein RF value Over
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