Technique / Proteomics and protein biochemistry / Protein electrophoresis

Protein electrophoresis buffer recipes and running condition

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	By binish on 11-Apr-2008 Can someone help me in telling me which protocol should i use for protein extract from leaves.Can someone give me the whole protocol for protein extraction? Thanks Rating: Excellent! Reply

By LU on 24-Aug-2006 My Western blot protoclos: Preparation of cell lysates From bacterial: 1.Spin down 1 ML overnight culture 2.add 100-200 ul BugBuster protein extraction reagent (Novagen cat. No. 70584) 3.Sonicate 1min 4. add 2x sample buffer to same lysis volume(or add sample buffer directly to the cell pallets) 5.Boiling 5 min Preparation of PVDF membrane: 1.Cut a piece of PVDF membrane(Bio-Rad Immun-Blot PVDF Membrane Cat. No. 162-0177) 2.Wet 30''-60" in Methanol 3.Transfer membrane to 1x Transfer buffer( Life gels LongLife Transfer Buffer 20x Cat.No. BG-168). untill to use. Using precast gels(Ready gel from Lifegels Cat. No.NH21-420 or BioRad cat. No.161-1104 ) 1. Assemble gel in gel tank(BioRad Mini-PROTEAN 3 Cell cat. No. 165-3301 165-3302) 2 prepare pretein 10-20 ug 3. Use 15 ul (Invitrogen SeeBlue Plus2 Pre-Stained Standard cat no. LC5925) as standard. 4.Run at 100V-150V(constant voltage) for 50 min-1.5 hour (must use Hepes-Tris running Buffer) Membrane Transfer: 1. pre wed the sponge, filter papers(Biorad cat. no.1703932) in 1X Transfer buffer 2.Assemble "sanwich" in BioRad transblot cat no.170-3930 170-3935) Black-sponges, filter paper-gel-membrane-filter paper-sponge-red 3.transfer at 200-250 mM(around 100V) for 1-2 hour with cold pack or pre-chilled buffer .Larger protein take longer time to transfer 4. when finished transfer, takemembrane out straightly if transfer buffer contain 20% methanol, (or soak in methanol again about 15") let it completely dry in RT.(over 30 min) 5.Wet membrane in methanol 15sec, and resin in dH2O. 6.Add in 5 % Bloking buffer(5 g dry milk BioRad cat.no.170-6404 in TBS-t0.1% tween20) and block overnight 4C or 1-2 hour RT Antibodies detection: 1.Incubator with primary antibody diluted in 1% Blocking buffer in TBS-t 0.1% tween20) for 1-2 hour RT 2. wash 3x 10 min with 0.1% Tween 20 in TBS-t 3.Incubate with Secondary antobody(Amersham NA934V) 1:10000 in 1% Blocking buffer 0.1%tween20 TBS-t for 45min-60min 4.wash 3X10 min with 0.1% tween20 in TBS-t. Wash with dH2O briefly before ECL. 5.Detect with Amersham ECL Plus kit (RPN 2132) Bring ECL plus kit to romm temperature. Make solution A:B 40:1 mix. Add 2ml to i membrane for 2 min. wrap with film for either film develop or camera picture. Do no let membrane dry. Strpping blot: 1 Rinse blot off with 0.1% Tween 20 in TBS. 2. Add Strpping buffer 37C 30 min(Pierce cat.no.21059) 3.Rinse blot off with 0.1% Tween 20 in TBS 4. Block about 1 hour with 5% blocking buffer/tween20. or overnight cold room. Rating: Excellent! Reply

	By canary on 12-Oct-2004 I am in trouble with the SDS loading buffer components. I don't know how to change them so that I can get stronger bands after Western blot. Rating: Good Reply

	By anila gill on 15-Jul-2003 please give me a buffer recipe for a pH 7.4 , ~ 10-4 mol/L buffer Rating: Good Reply

	By nmatta on 23-Nov-2002 I;m running leishmanial antigens, but I have problmes with de time, it is around 10-11 hours, of course at this time all the proteins are difunded. Im running a 120v and in average 30mA. I have tested all reagents, if someone can suggest someting Thanks a lot Rating: Good Reply