上海允麦生物科技有限公司
2013/2/27 13:53:15Preparation 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
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).
Anti-CD8/RBITC 罗丹明标记CD8抗体IgG |
Anti-AD7C/FITC 荧光素标记神经丝蛋白抗体IgG |
Anti-AD7C-NTP/NTP/AF /FITC 荧光素标记神经丝蛋白抗体(人)IgG |
Anti-AE3/FITC 荧光素标记阴离子交换蛋白3抗体IgG |
Anti-AEV/FITC 荧光素标记禽脑脊髓炎病毒抗体IgG |
Anti-AEBP1/FITC 荧光素标记脂肪细胞增强结合蛋白1抗体IgG |
Anti-AGER/FITC 荧光素标记晚期糖基化终末产物特异性受体抗体IgG |
Anti-AGEs/FITC 荧光素标记晚期糖基化终末产物抗体IgG |
Anti-Aggrecan/FITC 荧光素标记软骨蛋白聚糖/可聚蛋白多糖抗体IgG |
Anti-Agrin/FITC 荧光素标记聚集蛋白抗体IgG |
Anti-Agtr1/AT1a/FITC 荧光素标记血管组织血管紧张素Ⅱ1型受体抗体IgG |
Anti-AGER/Gold 金离子标记的晚期糖基化终末产物特异性受体抗体 |
Anti-Ah Receptor/FITC 荧光素标记芳香烃受体抗体IgG |
Anti-AHR/FITC 荧光素标记芳香烃受体抗体IgG |
Anti-AIF/FITC 荧光素标记Anti-AIF抗体IgG |
Anti-AIF/FITC 荧光素标记调亡诱导因子抗体IgG |
Anti-A/H1N1-M2 Human/Gold 胶体金标记兔抗人A型流感病毒H1N1-M2抗体IgG |
Anti-A/H1N1-M2 Human/Gold 胶体金标记兔抗人A型流感病毒H1N1-M2抗体IgG |
Anti-AIMP2/JTV1/FITC 荧光素标记抗AIMP2抗体IgG |
Anti-AK-1/FITC 荧光素标记腺苷酸激酶-1抗体IgG |