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罗丹明标记CD8抗体IgG相关产品信息

Preparation of cell lysates 

Collect cells （confluent T-25） by trypsinization and spin. 
Lyse the pellet with 100 µl lysis buffer on ice for 10 min. 
For 500,000 cells, lyse with 20 µl. 
Spin at 14,000 rpm （16,000 g） in an Eppendorf microfuge for 10 min at 4°C. 
Transfer the supernatant to a new tube and discard the pellet. 
Determine the protein concentration （Bradford assay, A280, or BCA） 
（We use the Bradford assay from Bio-Rad.） 
Take x µl （= y µg protein） and mix with x µl of 2x sample buffer. 
Boil for 5 min. 
Cool at RT for 5 min. 
Flash spin to bring down condensation prior to loading gel. 

B. Polyacrylamide gel （14.5 cm x 16.5 cm） 

Agarose plug: 
1% agarose dissolved in 1x Resolving gel buffer. 
（I make 50 ml, keep melting it as I need it, and re-adding water to maintain agarose conc.） 
Resolving gel: 24 ml of a 9% gel 
5.4 ml 40% acrylamide/bisacrylamide （29:1 mix） 
3 ml 8x Resolving gel buffer 
15.6 ml water 
12 µl TEMED 
60 µl 20% ammonium persulfate 
Stacking gel: 8 ml 
1 ml 40% acrylamide/bisacrylamide （29:1 mix） 
2 ml 4x Stacking gel buffer 
5 ml water 
8 µl TEMED 
21.6 µl 20% ammonium persulfate 

C. Preparation of gel 

Assemble the glass plates and spacers （1.5 mm thick）. 
Pour an agarose plug （1-2 mm）.